Vaccines Comprising Mycobacterium Leprae Polypeptides for the Prevention, Treatment, and Diagnosis of Leprosy

ABSTRACT

Compositions and methods for preventing, treating and detecting leprosy are disclosed. The compositions generally comprise polypeptides comprising one or more  Mycobacterium leprae  antigens as well as polynucleotides encoding such polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. patent application Ser. No.16/333,596, filed Mar. 15, 2019, which claims the benefit of PCTApplication No. PCT/US2017/051824, filed Sep. 15, 2017, which claims thebenefit of U.S. Patent Application No. 62/396,074, filed Sep. 16, 2016,all of which are hereby incorporated by reference herein in theirentirety, including all references and appendices cited therein, for allpurposes, as if fully set forth herein.

SUBMISSION OF SEQUENCE LISTING

The content of the following submission of a Sequence Listing XML isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing with file name:40-US-01_Sequence_Listing_ST26.XML, date created: Mar. 13, 2023, size:19,983 bytes).

BACKGROUND Technical Field

The present disclosure relates generally to compositions and methods forpreventing, treating and detecting leprosy in patients. Moreparticularly, the disclosure relates to compositions and methodscomprising Mycobacterium leprae antigens and fusion polypeptides, aswell as polynucleotides encoding such antigens and fusion polypeptides.

Description of the Related Arts

Leprosy (Hansen's disease) is an infectious peripheral neurologicaldisorder caused by Mycobacterium leprae. Nerve involvement in leprosypatients can present as sensory and/or motor neuron damage and canadvance to cause disability and disfigurement. Nerve damage likelyinvolves a complicated interplay of both host immunity and mycobacterialinfection-mediated events (1, 2). Although bacterial cure can beachieved by multidrug therapy (MDT), which the World Health Organization(WHO) provides free of charge for registered leprosy patients, leprosyremains as a public health problem in many regions. Declines in globalincidence prompted by the introduction of MDT and drive toward‘elimination’ as a global health problem by the year 2000 have nowlevelled off. More worryingly, it is widely believed that a large numberof cases go unreported (Smith, 2015 #4604). Recent new case incidencerates indicate that transmission continues and the disease is slowlyre-emerging in many regions that previously reported elimination.

The current pursuit of preventative measures against leprosy involvesprovision of MDT for patients or chemoprophylaxis within high riskpopulations. These strategies, however, are limited. Unlike drugtreatment, vaccines could be used to potentially provide active andsustained protection in both uninfected and infected individuals.Multibacillary (MB) leprosy patients present with many disseminated skinlesions and large bacterial burdens, indicating that the strong humoralimmune responses that they classically exhibit are not protective.Replication and dissemination of M. leprae is limited in paucibacillary(PB) leprosy patients, however, suggesting the potent cellular immuneresponse they develop is associated with limited or localized disease.In addition, despite presumed exposure to M. leprae, the vast majorityof healthy household contacts (HHC) of MB patients appear to developeffective immunity. Understanding the targets of the immune response ofthese individuals is likely the key to generating an effective vaccine.

By promoting a lasting adaptive immune response, a vaccine, unlike drugtreatment, has the potential to provide active and sustained protection.The current standard—and only administered-vaccine against M. leprae isthe BCG vaccine, originally developed for use in tuberculosis. Thepersistence of leprosy in countries where BCG is implemented suggestsits effectiveness is limited. (Goulart I M, Clin Vaccine Immunol 2008;15(1): 101-5.) The degree of protection afforded by BCG against leprosyhas varied dramatically between studies. Systematic meta-analysesindicate that BCG has a wide-ranging protective efficacy with an averagearound 50% and protection appears to be better against the MB than PBforms. (Setia M S et al., Lancet Infect Dis 2006; 6(3): 162-70; Merle CS, Expert review of vaccines. 2010; 9(2): 209-22) Furthermore, BCGvaccination has been shown to precipitate paucibacillarv (PB) leprosy insome instances, negating its limited usefulness.

M. leprae itself as an immunogen has been assessed in various trials,often to see if it can add to the protective effect of BCG. Large-scalestudies in Venezuela, Malawi and India testing the use of killed M.leprae in combination with BCG have been largely inconclusive, with widediscrepancies in results. (Convit J et al., Lancet 1992; 339(8791):446-50; Karonga Prevention Trial Group, Lancet 1996; 348(9019): 17-24)As a practical matter, production of a vaccine using killed M. lepraewould be enormously constrained by the difficulties associated with massproduction.

Accordingly, there remains a significant need for compositions andvaccines that can effectively prevent, treat and/or diagnose leprosy inhumans and other mammals. The present disclosure fulfills these needsand offers other related advantages.

BRIEF SUMMARY

The present disclosure provides compositions, kits and methods forpreventing, treating and detecting leprosy.

In one aspect the disclosure provides compositions comprising at leasttwo Mycobacterium leprae (M. leprae) antigens selected from the groupconsisting of ML2028, ML2055, and ML2380, or at least two M. lepraeantigens each having at least 90% amino acid sequence identity toML2028, ML2055, or ML2380. In some embodiments, the compositioncomprises ML2028 and ML2055; or an M. leprae antigen having at least 90%amino acid identity to ML2028 and an M. leprae antigen having at least90% amino acid identity to ML2055. In some embodiments, the compositioncomprises ML2028 and ML2380; or an M. leprae antigen having at least 90%amino acid identity to ML2028 and an M. leprae antigen having at least90% amino acid identity to ML2380. In some embodiments, the compositioncomprises ML2055 and ML2380; or an M. leprae antigen having at least 90%amino acid identity to ML2055 and an M. leprae antigen having at least90% amino acid identity to ML2380. In some embodiments, the compositioncomprises ML2028, ML2055, and ML2380; or an M. leprae antigen having atleast 90% amino acid identity to ML2028, an M. leprae antigen having atleast 90% amino acid identity to ML2055, and an M. leprae antigen havingat least 90% amino acid identity to ML2380. In some embodiments, thecomposition further comprises ML2531 or a M. leprae antigen having atleast 90% amino acid sequence identity to ML2531.

In another aspect the disclosure provides fusion polypeptides comprisingat least two Mycobacterium leprae (M. leprae) antigens selected from thegroup consisting of ML2028, ML2055, and ML2380, or at least two M.leprae antigens each having at least 90% amino acid sequence identity toML2028, ML2055, or ML2380. In some embodiments, the fusion polypeptidecomprises ML2028 and ML2055; or an M. leprae antigen having at least 90%amino acid identity to ML2028 and an M. leprae antigen having at least90% amino acid identity to ML2055. In some embodiments, the fusionpolypeptide comprises ML2028 and ML2380; or an M. leprae antigen havingat least 90% amino acid identity to ML2028 and an M. leprae antigenhaving at least 90% amino acid identity to ML2380. In some embodiments,the fusion polypeptide comprises ML2055 and ML2380; or an M. lepraeantigen having at least 90% amino acid identity to ML2055 and an M.leprae antigen having at least 90% amino acid identity to ML2380. Insome embodiments, the fusion polypeptide comprises ML2028, ML2055, andML2380; or an M. leprae antigen having at least 90% amino acid identityto ML2028, an M. leprae antigen having at least 90% amino acid identityto ML2055, and an M. leprae antigen having at least 90% amino acididentity to ML2380. In some embodiments, the fusion polypeptide furthercomprises M. leprae antigen ML2531. In some embodiments, the fusionpolypeptide comprises the sequence of SEQ ID NO: 12, or a sequencehaving 90% sequence identity thereto.

In another aspect, the disclosure provides isolated polynucleotidesencoding the fusion polypeptides of the disclosure.

In another aspect, the disclosure provides compositions comprising afusion polypeptide of the disclosure.

In some embodiments of the above aspects, ML2028 comprises the sequenceof SEQ ID NO: 2. In some embodiments, ML2028 comprises the sequence ofSEQ ID NO: 4. In some embodiments, ML2055 comprises the sequence of SEQID NO: 6. In some embodiments, ML2380 comprises the sequence of SEQ IDNO: 8. In some embodiments, ML2531 comprises the sequence of SEQ ID NO:10.

In some embodiments of compositions of the disclosure, the compositionfurther includes an immunostimulant. In some embodiments, theimmunostimulant is selected from the group consisting of aCpG-containing oligonucleotide, synthetic lipid A, MPL™, 3D-MPL™,saponins, saponin mimetics, AGPs, Toll-like receptor agonists, or acombination thereof. In some embodiments, the immunostimulant isselected from the group consisting of a TLR4 agonist, a TLR7/8 agonistand a TLR9 agonist. In some embodiments, the immunostimulant is selectedfrom the group consisting of GLA, CpG-containing oligonucleotide,imiquimod, gardiquimod and resiquimod.

In some embodiments the immunostimulant is GLA, having the followingstructure:

wherein R¹, R³, R⁵ and R⁶ are C₁₁-C₂₀ alkyl; and R² and R⁴ are C₉-C₂₀alkyl. In some embodiments. R¹, R³, R⁵ and W⁶ are C₁₁₋₁₄ alkyl; and R²and R⁴ are C₁₂₋₁₅ alkyl. In some embodiments, R¹, R³, W⁵ and W⁶ are C₁₁alkyl; and R² and W⁴ are C₁₃ alkyl. In some embodiments, R¹, R³, R⁵ andR⁶ are C₁₁ alkyl; and R² and R¹⁴ are C₉ alkyl.

In some embodiments, the immunostimulant has the following structure:

In some embodiments, the immunostimulant has the following structure:

In another aspect, the disclosure provides methods for stimulating animmune response against M. leprae in a mammal comprising administeringto a mammal in need thereof a composition of the disclosure. In someembodiments, the method further comprises administering to the mammal M.bovis BCG vaccine. In some embodiments, M. bovis BCG vaccine waspreviously administered to the mammal. In some embodiments, the mammalhas not been exposed to M. leprae. In some embodiments, the mammal hasbeen exposed to M. leprae. In some embodiments, the mammal is a humanhealthy household contact of a human identified as being infected withM. leprae. In some embodiments, the mammal has been infected by M.leprae. In some embodiments, the mammal exhibits signs or symptoms ofinfection by M. leprae.

In another aspect, the disclosure provides methods for stimulating animmune response against a tuberculosis-causing mycobacterium in a mammalcomprising administering to a mammal in need thereof a composition ofthe disclosure.

In another aspect, the disclosure provides methods for treating an M.leprae infection in a mammal, the method comprising administering to amammal having an M. leprae infection a composition of the disclosure. Insome embodiments, the method further comprises administering to themammal one or more chemotherapeutic agents. In some embodiments, the oneor more chemotherapeutic agents comprise one or more agents selectedfrom the group consisting of dapsone, rifampicin, clofazimine,ofloxacin, minocycline, gatifloxacin, linezolid, and PA 824. In someembodiments, the mammal is first administered one or morechemotherapeutic agents over a period of time and subsequentlyadministered the composition. In some embodiments, the mammal is firstadministered the composition and subsequently administered one or morechemotherapeutic agents over a period of time. In some embodiments,administration of the one or more chemotherapeutic agents and thecomposition is concurrent. In some embodiments, the method furthercomprises administering the composition to the mammal one or moresubsequent times. In some embodiments, the method further comprisesadministering to the mammal M. bovis BCG vaccine. In some embodiments,M. bovis BCG vaccine was previously administered to the mammal. In someembodiments, the mammal does not exhibit signs or symptoms of infectionby M. leprae. In some embodiments, the mammal has indeterminate ortuberculoid presentation. In some embodiments, the mammal haspaucibacillary leprosy. In some embodiments, the mammal hasmultibacillary leprosy. In some embodiments, the mammal has lepromatousleprosy. In some embodiments, the mammal has borderline lepromatousleprosy. In some embodiments, the mammal has mid-borderline leprosy. Insome embodiments, the mammal has borderline tuberculoid leprosy. In someembodiments, the mammal has tuberculoid leprosy. In some embodiments,the mammal is infected with a multidrug resistant M. leprae.

In another aspect, the disclosure provides methods for reducing the timecourse of chemotherapy against an M. leprae infection, the methodcomprising administering to a mammal having an M. leprae infection acomposition of the disclosure in conjunction with the chemotherapy,where the composition induces an immune response against M. leprae,thereby providing for a reduced time course of the chemotherapy againstan M. leprae infection. In some embodiments, the time course ofchemotherapy is shortened to no more than about 3 months, about 5months, or about 7 months.

In some embodiments of the methods of the disclosure, the mammal is ahuman.

In another aspect, the disclosure provides a method for detecting M.leprae infection in a biological sample, comprising: (a) contacting abiological sample with a polypeptide (including a fusion polypeptide) asdescribed herein; and (b) detecting in the biological sample thepresence of antibodies that bind to the fusion polypeptide, therebydetecting M. leprae infection in a biological sample. Any suitablebiological sample type may be analyzed by the method, illustrativeexamples of which may include, for example, sera, blood, saliva, skin,and nasal secretion.

In certain embodiments of the disclosed diagnostic methods, thepolypeptide (including a fusion polypeptide) is bound to a solidsupport. Accordingly, the present disclosure further provides diagnosticreagents comprising a polypeptide (including a fusion polypeptide) asdescribed herein, immobilized on a solid support.

Diagnostic kits for detecting M. leprae infection in a biological sampleare also provided, generally comprising a polypeptide (including afusion polypeptide) as described herein and a detection reagent. It willbe understood that the kit may employ a polypeptide (including a fusionpolypeptide) of the disclosure in any of a variety of assay formatsknown in the art, including, for example, a lateral flow test stripassay, a dual path platform (DPP) assay and an ELISA assay. These kitsand compositions of the disclosure can offer valuable point of carediagnostic information. Furthermore, the kits and compositions can alsobe advantageously used as test-of-cure kits for monitoring the status ofinfection in an infected individual over time and/or in response totreatment.

Treatment kits for treating an M. leprae infection in a mammal are alsoprovided, generally comprising a composition of the disclosure.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present disclosure. These and other aspects of thepresent disclosure will become apparent upon reference to the followingdetailed description and attached drawings. All references disclosedherein are hereby incorporated by reference in their entirety as if eachwas incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Immunization with recombinant antigens formulated in GLA-SEreduce M. leprae burden. Mice were injected s.c. with antigens/GLA-SE atbiweekly intervals, for a total of 3 immunizations. One month after thelast immunization mice were infected with 1×10⁴ M. leprae in each foot,and bacterial burdens determined 12 months later. Results are shown asmean and SE. Mann-Whitney test was used to calculate p-values betweeneach group; n=6 per group. FIG. 1A shows the antigens, ML2028, ML2055and ML2380, administered individually compared to the sham treatment andheat-killed M. leprae. FIG. 1B shows the results of administering theantigens in combination. FIG. 1C shows the negative results fromimmunization with the antigens ML0276 and ML46F.

FIG. 2 : Immunological recognition of each component is retained inchimeric fusion protein ML89. Mice were injected s.c. withantigens/GLA-SE at biweekly intervals, for a total of 3 immunizations.Serum and spleens were collected one month after the third immunization.In FIG. 2 , antigen-specific serum IgG, IgG1 and IgG2a titers inresponse to ML89 immunization were determined by ELISA for ML89 (FIG.2A), ML2028 (FIG. 2B), ML2055 (FIG. 2C), and ML2380 (FIG. 2D).

FIG. 3 : Prior BCG priming does not alter the induction of anti-M.leprae responses by ML89 immunization. Single cell suspensions wereprepared from each spleen and cultured with 10 μg/ml protein. Culturesupematants were collected and IFN γ content determined by ELISA.Results are shown as mean and SE; n=3 per group. Data are representativeof two independent experiments. The results show recall responsefollowing ML89 immunization (with and without BCG priming) to thefollowing antigens in FIGS. 3A-3E: ML89 (FIG. 3A), BCG lysate (FIG. 3B),purified protein derivative (PPD) (FIG. 3C), M. leprae cell sonicate(MLCS) (FIG. 3D), and cell wall antigen (CWA) (FIG. 3E).

FIG. 4 : Immunization with ML89/GLA-SE reduces M. leprae burden. Micewere injected s.c. with antigens/GLA-SE at biweekly intervals, for atotal of 3 immunizations. One month after the last immunization micewere infected with 1×10⁴ M. leprae in each foot, and bacterial burdensdetermined 12 months later. Results were generated in 2 independentlaboratories and are shown as mean and SE. Mann-Whitney test was used tocalculate p-values between each group; n=6 per group. FIG. 4Ademonstrates an 85% reduction of M. leprae bacterial burden. FIG. 4Bshows that repeated ML89 administration still protects against M. lepraegrowth.

FIG. 5 : Immunization with ML89/GLA-SE delays M. leprae-induced nervedamage. FIG. 5A shows that untreated armadillos demonstrated early onsetof defects, while onset in ML89-immunized armadillos was comparativelydelayed. FIG. 5B shows that ML89-immunized armadillos had lowerincidence of sustained nerve conduction defects, whereas BCG-immunizedarmadillos developed more rapid onset of severe damage relative to bothcontrol and ML-89 treated animals. FIG. 5C shows the proportion ofanimals displaying normal, borderline, and abnormal nerve conduction incontrol, BCG, and LepVax-treated armadillos.

BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS

SEQ ID NO: 1 is a nucleic acid sequence encoding the ML2028 antigenpolypeptide of SEQ ID NO: 2.

SEQ ID NO: 2 is an amino acid sequence of Mycobacterium leprae ML2028antigen (diacylglycerol acyltransferase; NCBI Reference Sequence:WP_010908679.1).

SEQ ID NO: 3 is a nucleic acid sequence encoding the ML202839-327polypeptide of SEQ ID NO: 4.

SEQ ID NO: 4 is an amino acid sequence of the mature chain withoutsignal sequence of ML2028 antigen from Mycobacterium leprae(ML202839-327).

SEQ ID NO: 5 is a nucleic acid sequence encoding the ML2055 antigenpolypeptide of SEQ ID NO: 6.

SEQ ID NO: 6 is an amino acid sequence of ML2055 antigen fromMycobacterium leprae (alanine and proline-rich secreted protein Apa;NCBI Reference Sequence: WP_010908692.1).

SEQ ID NO: 7 is a nucleic acid sequence encoding the ML2380 antigenpolypeptide of SEQ ID NO: 8.

SEQ ID NO: 8 is an amino acid sequence of ML2380 antigen fromMycobacterium leprae (hypothetical protein; NCBI Reference Sequence:WP_010908863.1).

SEQ ID NO: 9 is a nucleic acid sequence encoding the ML2531 antigenpolypeptide of SEQ ID NO: 10.

SEQ ID NO: 10 is an amino acid sequence of ML2531 antigen fromMycobacterium leprae (ESAT-6-like protein EsxR: NCBI Reference Sequence:WP_010908945.1).

SEQ ID NO: 11 is a nucleic acid sequence encoding the LEP-F1 fusionpolypeptide of SEQ ID NO: 12.

SEQ ID NO: 12 is an amino acid sequence of the LEP-F1 fusionpolypeptide.

DETAILED DESCRIPTION

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of molecular biology, recombinantDNA, and chemistry, which are within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., MolecularCloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold SpringHarbor Laboratory Press: (1989); DNA Cloning, Volumes I and II (D. N.Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);Mullis et al., U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B.D. Hames & S. J. Higgins eds. 1984); B. Perbal, A Practical Guide ToMolecular Cloning (1984); the treatise, Methods In Enzymology (AcademicPress, Inc., N.Y.); and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Maryland (1989).

As noted above, the present disclosure is generally directed tocompositions and methods for preventing, treating and detecting leprosy.The compositions of the disclosure include, for example, polypeptidesincluding fusion polypeptides that comprise various immunogenic portionsof Mycobacterium leprae (M. leprae) proteins, wherein the portions andvariants preferably retain substantially the same or similar immunogenicproperties as a corresponding full length M. leprae protein.Immunization strategies using compositions of the disclosure can beapplied to the in vivo protection against, for example, infection by M.leprae, which is the causative agent of leprosy in humans andarmadillos. The present disclosure also contemplates, in otherembodiments, using the polypeptides including fusion polypeptidesdescribed herein in methods of treating mammals having an M. lepraeinfection. The present disclosure also contemplates, in otherembodiments, using the polypeptides including fusion polypeptidesdescribed herein in diagnostic applications, including, but not limitedto, diagnosis and whole blood assays, preferably in a format amenable toproviding rapid, point of care diagnostic results, such as a lateralflow assay or a dual path platform assay.

M. Leprae Antigens and Fusion Polypeptides and Uses Therefor

In a general aspect, the present disclosure provides M. leprae antigens,as described herein, including fusion polypeptides and compositionscontaining the same.

In some embodiments the disclosure provides compositions comprising atleast two Mycobacterium leprae (M. leprae) antigens selected from thegroup consisting of ML2028, ML2055, and ML2380, or at least two M.leprae antigens each having at least 90% amino acid sequence identity toML2028, ML2055, or ML2380. In some embodiments, the compositioncomprises ML2028 and ML2055; or an M. leprae antigen having at least 90%amino acid identity to ML2028 and an M. leprae antigen having at least90% amino acid identity to ML2055. In some embodiments, the compositioncomprises ML2028 and ML2380; or an M. leprae antigen having at least 90%amino acid identity to ML2028 and an M. leprae antigen having at least90% amino acid identity to ML2380. In some embodiments, the compositioncomprises ML2055 and ML2380; or an M. leprae antigen having at least 90%amino acid identity to ML2055 and an M. leprae antigen having at least90% amino acid identity to ML2380. In some embodiments, the compositioncomprises ML2028, ML2055, and ML2380; or an M. leprae antigen having atleast 90% amino acid identity to ML2028, an M. leprae antigen having atleast 90% amino acid identity to ML2055, and an M. leprae antigen havingat least 90% amino acid identity to ML2380. In some embodiments, thecomposition further comprises ML2531 or an M. leprae antigen having atleast 90% amino acid sequence identity to ML2531.

In some embodiments the disclosure provides fusion polypeptidescomprising at least two Mycobacterium leprae (M. leprae) antigensselected from the group consisting of ML2028, ML2055, and ML2380, or atleast two M. leprae antigens each having at least 90% amino acidsequence identity to ML2028, ML2055, or ML2380. In some embodiments, thefusion polypeptide comprises ML2028 and ML2055; or an M. leprae antigenhaving at least 90% amino acid identity to ML2028 and an M. lepraeantigen having at least 90% amino acid identity to ML2055. In someembodiments, the fusion polypeptide comprises ML2028 and ML2380; or anM. leprae antigen having at least 90% amino acid identity to ML2028 andan M. leprae antigen having at least 90% amino acid identity to ML2380.In some embodiments, the fusion polypeptide comprises ML2055 and ML2380;or an M. leprae antigen having at least 90% amino acid identity toML2055 and an M. leprae antigen having at least 90% amino acid identityto ML2380. In some embodiments, the fusion polypeptide comprises ML2028.ML2055, and ML2380; or an M. leprae antigen having at least 90% aminoacid identity to ML2028, an M. leprae antigen having at least 90% aminoacid identity to ML2055, and an M. leprae antigen having at least 90%amino acid identity to ML2380. In some embodiments, the fusionpolypeptide further comprises M. leprae antigen ML2531. In someembodiments, the fusion polypeptide comprises the sequence of SEQ ID NO:12, or a sequence having 90% sequence identity thereto.

In some embodiments, compositions comprising antigens and fusionpolypeptides described herein can generate an immune response or aneffective immune response to M. leprae. The immune response may have oneor more of the following characteristics: 1) a reduction in bacterialburden in immunized hosts upon challenge with an M. leprae infection; 2)secretion of IFNγ in vitro spleen cell cultures from mice immunized withthe compositions of the disclosure upon incubation with the matchedfusion polypeptide or individual antigens of the fusion polypeptide; 3)IFNγ secretion in vitro spleen cell cultures from mice immunized withthe compositions of the disclosure following incubation with crude M.leprae, 4) generation of antigen-specific multifunctional Th1 cells, forexample CD4 T cells that produce multiple cytokines indicative of a Th1phenotype such as the combined production of IFNγ, TNF and IL-2 or IFNγand TNF; or 5) improvement or enhancement of the immune recognition ofone or more antigen(s), when presented in the context of a fusionpolypeptide, as measured for example by the secretion of cytokines suchγIFN, or the titer of presence of antibodies or cellular responses tothe antigen. Methods for testing one or more of the above immuneresponses are known in the art and are described in detail in Examples.

Different M. leprae antigens in the fusion polypeptides may be arrangedin the fusion polypeptide in any order. For example, any particularpolypeptide of the fusion polypeptide may be located towards theC-terminal end of the fusion polypeptide or the N-terminal end of thepolypeptide or in the center of the fusion polypeptide {i.e., located inbetween at least two other polypeptides in the fusion polypeptide).Different M. leprae antigens may be linked by a linker sequence of anylength (e.g., 2-20 amino acids).

In one embodiment, the fusion polypeptide consists of four M. lepraeantigens: ML2531 (ESAT-6-like protein EsxR), ML2380 (hypotheticalprotein), ML2055 (cell surface protein associated with virulence), andML202839-327 (antigen 85B, mature chain without signal sequence). Thefull native sequence of ML2531, ML2380, ML2055 are present, whileML202839-327 represents the mature chain without the signal sequenceresidues 1 through 38.

There is a two-residue linker sequence inserted between each of theantigens to improve expression and recovery. The resulting 831 aminoacid fusion protein has a predicted molecular weight of 89,062 Da. Thepolynucleotide sequence encoding the fusion polypeptide is SEQ ID NO:11, and the amino acid sequence of the fusion polypeptide is SEQ ID NO:12. Such fusion polypeptide may be referred to as LEP-F1, ML89, orLepVax.

A schematic of one embodiment of the fusion polypeptide is below.

ML2531 ML2380 ML2055 ML2028₃₉₋₃₂₇ 10 kDa 17 kDa 30 kDa 31 kDa

As used herein, the term “polypeptide” or “protein” encompasses aminoacid chains of any length, including full length proteins, wherein theamino acid residues are linked by covalent bonds. An antigen is apolypeptide comprising an immunogenic portion of a M. leprae polypeptideor protein and may consist solely of an immunogenic portion, may containtwo or more immunogenic portions and/or may contain additionalsequences. The additional sequences may be derived from a native M.leprae polypeptide or protein or may be heterologous, and suchheterologous sequences may (but need not) be immunogenic.

An “isolated polypeptide” is one that is removed from its originalenvironment. For example, a naturally-occurring protein is isolated ifit is separated from some or all of the coexisting materials in thenatural system. Preferably, such polypeptides are at least about 90%pure, more preferably at least about 95% pure and most preferably atleast about 99% pure. One of ordinary skill in the art would appreciatethat antigenic polypeptide fragments could also be obtained from thosealready available in the art. Polypeptides of the disclosure,antigenic/immunogenic fragments thereof, and other variants may beprepared using conventional recombinant and/or synthetic techniques.

The M. leprae antigens used in a fusion polypeptide of the presentdisclosure can be full length, substantially full length polypeptides,or variants thereof as described herein. Alternatively, a fusionpolypeptide or composition of the disclosure can comprise or consist ofimmunogenic portions or fragments of a full length M. lepraepolypeptide, or variants thereof.

In certain embodiments, an immunogenic portion of a M. lepraepolypeptide is a portion that is capable of eliciting an immune response(i.e., cellular and/or humoral) in a presently or previously M.leprae-infected patient (such as a human or a mammal (e.g., anarmadillo)) and/or in cultures of spleen cells, lymph node cells orperipheral blood mononuclear cells (PBMC) isolated from presently orpreviously M. leprae-infected individuals. The cells in which a responseis elicited may comprise a mixture of cell types or may contain isolatedcomponent cells (including, but not limited to, T-cells, NK cells,macrophages, monocytes and/or B cells). In a particular embodiment,immunogenic portions of a fusion polypeptide of the disclosure arecapable of inducing T-cell proliferation and/or a predominantly Th1-typecytokine response (e.g., IL-2, IFN-γ, and/or TNFα production by T-cellsand/or NK cells, and/or IL-12 production by monocytes, macrophagesand/or B cells). Immunogenic portions of the polypeptides describedherein may generally be identified using techniques known to those ofordinary skill in the art, including the representative methodssummarized in Paul, Fundamental Immunology, 5th ed., Lippincott Williams& Wilkins, 2003 and references cited therein. Such techniques includescreening fusion polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in animmunoassay, and do not react detectably with unrelated proteins). Suchantisera and antibodies may be prepared as described herein and usingwell-known techniques.

Immunogenic portions of an M. leprae polypeptide can be essentially anylength; provided they retain one or more of the immunogenic regions thatare responsible for or contribute to the in vivo protection providedagainst leprosy by one or more antigens of fusion polypeptides of thedisclosure, as disclosed herein. In one embodiment, the ability of animmunogenic portion to react with antigen-specific antisera may beenhanced or unchanged, relative to the native protein, or may bediminished by less than 50%, and preferably less than 20%, relative tothe native protein. Illustrative portions will generally be at least 10,15, 25, 50, 150, 200, 250, 300, or 350 amino acids in length, or more,up to and including full length M. leprae polypeptide.

In some embodiments, a M. leprae antigen described herein includesML2028, ML2055, ML2380, and ML2531. In some embodiments, these M. lepraeantigens include any naturally occurring variants.

As would be recognized by the skilled artisan, a composition of thedisclosure may also comprise one or more polypeptides that areimmunologically reactive with T cells and/or antibodies generatedagainst a polypeptide of the disclosure, particularly a polypeptidehaving an amino acid sequence disclosed herein, or to an immunogenicfragment or variant thereof. In a specific embodiment, the polypeptideis a fusion polypeptide, as described herein.

As noted, in various embodiments of the present disclosure, fusionpolypeptides generally comprise at least an immunogenic portion orvariant of the M. leprae polypeptides described herein. In someinstances, preferred immunogenic portions will be identified that have alevel of immunogenic activity greater than that of the correspondingfull-length polypeptide, e.g., having greater than about 100% or 150% ormore immunogenic activity. In particular embodiments, the immunogenicityof the fill-length fusion polypeptide will have additive, or greaterthan additive immunogenicity contributed by of each of theantigenic/immunogenic portions contained therein.

In another aspect, fusion polypeptides of the present disclosure maycontain multiple copies of polypeptide fragments, repeats of polypeptidefragments, or multimeric polypeptide fragments, includingantigenic/immunogenic fragments, such as M. leprae polypeptidescomprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morecontiguous fragments of aM leprae polypeptide, in any order, andincluding all lengths of a polypeptide composition set forth herein, orthose encoded by a polynucleotide sequence set forth herein.

In some embodiments, the ML2028 antigen comprises the sequence of SEQ IDNO: 2 or SEQ ID NO: 4, or a sequence having at least 90% identity (e.g.,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%) toSEQ ID NO: 2 or to SEQ ID NO: 4. In some embodiments, the ML2055 antigencomprises the sequence of SEQ ID NO: 6, or a sequence having at least90% identity (e.g., at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) to SEQ ID NO: 6. In some embodiments, the ML2380antigen comprises the sequence of SEQ ID NO: 8, or a sequence having atleast 90% identity (e.g., at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%) to SEQ ID NO: 8. In some embodiments, theML2531 antigen comprises the sequence of SEQ ID NO: 10, or a sequencehaving at least 90% identity (e.g., at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%) to SEQ ID NO: 10.

In another aspect, the disclosure provides a fusion polypeptidecomprising, consisting of, or consisting essentially of the amino acidsequence set forth in SEQ ID NO: 12, or a sequence having at least 90%identity thereto (e.g., at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity thereto).

In yet another aspect, the present disclosure provides fusionpolypeptides comprising one or more variants of the M. leprae antigensdescribed herein. Polypeptide variants generally encompassed by thepresent disclosure will typically exhibit at least about 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or moreidentity (determined as described below), along its length, to apolypeptide sequence set forth herein.

In other related embodiments, a polypeptide “variant,” includespolypeptides that differ from a native protein in one or moresubstitutions, deletions, additions and/or insertions, such that thedesired immunogenicity of the variant polypeptide is not substantiallydiminished relative to a native polypeptide.

For example, certain variants of the disclosure include polypeptides ofthe disclosure that have been modified to replace one or more cysteineresidues with alternative residues. Such polypeptides are referred tohereinafter as cysteine-modified polypeptides or cysteine-modifiedfusion polypeptides. Preferably, the modified polypeptides retainsubstantially the same or similar immunogenic properties as thecorresponding unmodified polypeptides. In a more specific embodiment,cysteine residues are replaced with serine residues because of thesimilarity in the spatial arrangement of their respective side chains.However, it will be apparent to one skilled in the art that any aminoacid that is incapable of interchain or intrachain disulfide bondformation can be used as a replacement for cysteine. When all orsubstantially all of the cysteine residues in a polypeptide or fusionpolypeptide of this disclosure are replaced, the resultingcysteine-modified variant may be less prone to aggregation and thuseasier to purify, more homogeneous, and/or obtainable in higher yieldsfollowing purification.

In one embodiment, the ability of a variant to react withantigen-specific antisera may be enhanced or unchanged, relative to thenative protein, or may be diminished by less than 50%, and preferablyless than 20%, relative to a corresponding native or controlpolypeptide. In a particular embodiment, a variant of an M. lepraepolypeptide is one capable of providing protection against M. lepraeinfection.

In particular embodiments, a fusion polypeptide of the presentdisclosure comprises at least 1, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, or at least 10or more substitutions, deletions, additions and/or insertions within aM. leprae polypeptide, where the fusion polypeptide is capable ofproviding protection against an M. leprae infection.

In related embodiments, a fusion polypeptide of the present disclosurecomprises at least 1, at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, or at least 10 or moresubstitutions, deletions, additions and/or insertions within a M. lepraepolypeptide, where the fusion polypeptide is capable of serodiagnosis ofM. leprae.

In many instances, a variant will contain conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. As described above, modifications may be made in thestructure of the polynucleotides and polypeptides of the presentdisclosure and still obtain a functional molecule that encodes a variantor derivative polypeptide with desirable characteristics, e.g., withimmunogenic characteristics. When it is desired to alter the amino acidsequence of a polypeptide to create an equivalent, or even an improved,immunogenic variant or portion of a polypeptide of the disclosure, oneskilled in the art will typically change one or more of the codons ofthe encoding DNA sequence according to Table 1.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated that various changes may bemade in the peptide sequences of the disclosed compositions, orcorresponding DNA sequences which encode said peptides withoutappreciable loss of their biological utility or activity.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAG GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gin Q CAA CAG Arginine Arg RAGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU ThreonineThr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics(Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within +2is preferred, those within +1 are particularly preferred, and thosewithin +0.5 are even more particularly preferred. It is also understoodin the art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1) serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

Amino acid substitutions may further be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity and/orthe amphipathic nature of the residues. For example, negatively chargedamino acids include aspartic acid and glutamic acid; positively chargedamino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine,isoleucine and valine: glycine and alanine: asparagine and glutamine;and serine, threonine, phenylalanine and tyrosine. Other groups of aminoacids that may represent conservative changes include: (1) ala, pro,gly, glu, asp, gin, asn, ser, thr: (2) cys, ser, tyr, thr: (3) val, ile,leu, met, ala, phe: (4) lys, arg, his; and (5) phe, tyr, trp, his. Avariant may also, or alternatively, contain nonconservative changes. Ina preferred embodiment, variant polypeptides differ from a nativesequence by substitution, deletion or addition of five amino acids orfewer. Variants may also (or alternatively) be modified by, for example,the deletion or addition of amino acids that have minimal influence onthe immunogenicity, secondary structure and hydropathic nature of thepolypeptide.

As noted above, polypeptides may comprise a signal (or leader) sequenceat the N-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-Histidine tag (6XHis), GST, MBP, TAP/TAG, FLAG epitope, MYCepitope, V5 epitope, VSV-G epitope, etc.), or to enhance binding of thepolypeptide to a solid support. For example, a polypeptide may beconjugated to an immunoglobulin Fc region.

When comparing polynucleotide or polypeptide sequences, two sequencesare said to be “identical” if the sequence of nucleotides or amino acidsin the two sequences is the same when aligned for maximumcorrespondence, as described below. Comparisons between two sequencesare typically performed by comparing the sequences over a comparisonwindow to identify and compare local regions of sequence similarity. A“comparison window” as used herein, refers to a segment of at leastabout 20 contiguous positions, usually 30 to about 75, 40 to about 50,in which a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned.

Alignment of sequences for comparison may be conducted using, forexample, the Megalign program in the Lasergene suite of bioinformaticssoftware (DNASTAR, Inc., Madison, WI), using default parameters. Thisprogram embodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358: HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-″645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA:Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153: Myers, E. W.and Muller W. (1988) CABIOS 4: 11-17: Robinson, E. D. (1971) Comb. Theor11:105: Santou, N. Nes, M. (1987) MoL Biol. Evol. 4:406-425: Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, CA;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

Alternatively, alignment of sequences for comparison may be conducted bythe local identity algorithm of Smith and Waterman (1981) Add. APL. Math2:482, by the identity alignment algorithm of Needleman and Wunsch(1970) J. MoL Biol. 48:443, by the search for similarity methods ofPearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, BLAST,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.

One example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nucl. AcidsRes. 25:3389-3402 and Altschul ct al. (1990) J. Mol. Biol. 215:403-410,respectively. BLAST and BLAST 2.0 can be used, for example with theparameters described herein, to determine percent sequence identity forthe polynucleotides and polypeptides of the disclosure. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. In one illustrative example,cumulative scores can be calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix can be used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W. Tand X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparisonof both strands.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e., the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Therefore, as noted above, the present disclosure encompassespolynucleotide and polypeptide sequences having substantial identity tothe sequences disclosed herein, for example those comprising at least70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequenceidentity compared to a polynucleotide or polypeptide sequence of thisdisclosure (e.g., as set out in SEQ ID NOs: 1-12) using the methodsdescribed herein, (e.g., BLAST analysis using standard parameters, asdescribed below). One skilled in this art will recognize that thesevalues can be appropriately adjusted to determine corresponding identityof proteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning andthe like. Furthermore, it would be understood by of ordinary skill inthe art that fusion polypeptides of the present disclosure may compriseat least 2, at least 3, or at least 4 or more antigenic/immunogenicportions or fragments of a polypeptide comprising at least 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identityto a M. leprae polypeptide that is capable of providing protectionagainst M. leprae infection, or serodiagnosis of M. leprae.

In another aspect of the disclosure, fusion polypeptides are providedthat comprise at least an immunogenic portion of a polypeptide andfurther comprise a heterologous fusion partner, as well aspolynucleotides encoding such fusion polypeptides. For example, in oneembodiment, a fusion polypeptide comprises one or more immunogenicportions or fragments of a M. leprae polypeptide and one or moreadditional immunogenic M. leprae sequences, which are joined via apeptide linkage into a single amino acid chain.

In another embodiment, a fusion polypeptide may comprise multiple M.leprae antigenic portions. In some embodiments, at least one of theportions in the fusion polypeptide is from ML2028, ML2055, or ML2380. Insome embodiments, an immunogenic portion is a portion of an antigen thatreacts with blood samples from M. leprae-infected individuals (i.e. anepitope is specifically bound by one or more antibodies and/or T-cellspresent in such blood samples.

In certain embodiments, a fusion polypeptide may further comprise atleast one heterologous fusion partner having a sequence that assists inproviding T helper epitopes (an immunological fusion partner),preferably T helper epitopes recognized by humans, or that assists inexpressing the protein (an expression enhancer) at higher yields thanthe native recombinant protein. Certain preferred fusion partnersinclude both immunological and expression-enhancing fusion partners.Other fusion partners may be selected so as to increase the solubilityof the protein or to enable the protein to be targeted to desiredintracellular compartments. Still further fusion partners includeaffinity tags, such as V5, 6XHIS, MYC, FLAG, and GST, which facilitatepurification of the protein. It would be understood by one havingordinary skill in the art that those unrelated sequences may, but neednot, be present in a fusion polypeptide used in accordance with thepresent disclosure. In another particular embodiment, an immunologicalfusion partner comprises an amino acid sequence derived from the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292 (1986)). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798 (1992)). Within a particularembodiment, a repeat portion of LYTA may be incorporated into a fusionprotein. A repeat portion is found in the C-terminal region starting atresidue 178. A more particular repeat portion incorporates residues188-305.

Fusion sequences may be joined directly (i.e., with no intervening aminoacids) or may be joined by way of a linker sequence (e.g., Gly-Cys-Gly)that does not significantly diminish the immunogenic properties of thecomponent polypeptides. The polypeptides forming the fusion protein aretypically linked C-terminus to N-terminus, although they can also belinked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminusto C-terminus. The polypeptides of the fusion protein can be in anyorder. Fusion polypeptides or fusion proteins can also includeconservatively modified variants, polymorphic variants, alleles,mutants, subsequences, interspecies homologs, and immunogenic fragmentsof the antigens that make up the fusion protein.

Fusion polypeptides may generally be prepared using standard techniques,including recombinant technology, chemical conjugation and the like. Forexample, DNA sequences encoding the polypeptide components of a fusionmay be assembled separately, and ligated into an appropriate expressionvector. The 3′ end of the DNA sequence encoding one polypeptidecomponent is ligated, with or without a peptide linker, to the 5′ end ofa DNA sequence encoding the second polypeptide component so that thereading frames of the sequences are in frame. This permits translationinto a single fusion polypeptide that retains or in some cases exceedsthe biological activity of the component polypeptides.

A peptide linker sequence may be employed to separate the fusioncomponents by a distance sufficient to ensure that each polypeptidefolds into its desired secondary and/or tertiary structures. Such apeptide linker sequence may be incorporated into the fusion polypeptideusing standard techniques well known in the art. Suitable peptide linkersequences may be chosen, for example, based on one or more of thefollowing factors: (1) their ability to adopt a flexible extendedconformation; (2) their inability to adopt a secondary structure thatcould interact with functional epitopes on the first and secondpolypeptides; and (3) the lack of hydrophobic or charged residues thatmight react with the polypeptide functional epitopes. Certain preferredpeptide linker sequences contain Gly. Asn and Ser residues. Other nearneutral amino acids, such as Thr and Ala may also be used in the linkersequence. Amino acid sequences which may be usefully employed as linkersinclude those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphyet al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. Nos.4,935,233 and 4,751,180. The linker sequence may generally be from 1 toabout 50 amino acids in length. Linker sequences are not required whenthe first and second polypeptides have non-essential N-terminal aminoacid regions that can be used to separate the functional domains andprevent steric interference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

In addition to recombinant fusion polypeptide expression, M. lepraepolypeptides, immunogenic portions, variants and fusions thereof may begenerated by synthetic or recombinant means. Synthetic polypeptideshaving fewer than about 100 amino acids, and generally fewer than about50 amino acids, may be generated using techniques well known to those ofordinary skill in the art. For example, such polypeptides may besynthesized using any of the commercially available solid-phasetechniques, such as the Merrifield solid-phase synthesis method, whereamino acids are sequentially added to a growing amino acid chain(Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963). Equipment forautomated synthesis of polypeptides is commercially available fromsuppliers such as Perkin Elmer/Applied BioSystems Division, Foster City,CA, and may be operated according to the manufacturer's instructions.Thus, for example, M. leprae antigens, or portions thereof, may besynthesized by this method.

Recombinant polypeptides containing portions and/or variants of a nativeM. leprae polypeptide may be readily prepared from a DNA sequenceencoding the antigen, using well known and established techniques. Inparticular embodiments, a fusion polypeptide comprising M. lepraeantigens may be readily prepared from a DNA sequence encoding the clonedfused antigens. For example, supematants from suitable host/vectorsystems which secrete recombinant protein into culture media may befirst concentrated using a commercially available filter. Followingconcentration, the concentrate may be applied to a suitable purificationmatrix such as an affinity matrix, a size exclusion chromatographymatrix or an ion exchange resin.

Alternatively, any of a variety of expression vectors known to those ofordinary skill in the art may be employed to express recombinantpolypeptides of this disclosure. Expression may be achieved in anyappropriate host cell that has been transformed or transfected with anexpression vector containing a polynucleotide that encodes a recombinantpolypeptide. Preferably, the host cells are E. coli, yeast, an insectcell line (such as Spodoptera or Trichoplusia) or a mammalian cell line,including (but not limited to) CHO, COS, HEK-293T and NS-1. The DNAsequences expressed in this manner may encode naturally occurringproteins, and fusion proteins comprising M. leprae antigens, such asthose described herein, portions thereof, and repeats or other variantsof such proteins. Expressed fusion polypeptides of this disclosure aregenerally isolated in substantially pure form. Preferably, the fusionpolypeptides are isolated to a purity of at least 80% by weight, morepreferably, to a purity of at least 95% by weight, and most preferablyto a purity of at least 99% by weight. In general, such purification maybe achieved using, for example, the standard techniques of ammoniumsulfate fractionation. SDS-PAGE electrophoresis, and affinitychromatography.

Regardless of the method of preparation, the polypeptides or fusionpolypeptides produced as described above are preferably immunogenic. Incertain embodiments, for example, the polypeptides (or immunogenicportions thereof) are capable of eliciting an immune response incultures of lymph node cells and/or peripheral blood mononuclear cells(PBMC) isolated from presently or previously M. leprae—infectedindividuals. More specifically, in certain embodiments, the antigens,and immunogenic portions thereof, have the ability to induce T-cellproliferation and/or to elicit a dominantly Th1-type cytokine response(e.g., IL-2, IFN-γ, and/or TNF-α production by T-cells and/or NK cells;and/or IL-12 production by monocytes, macrophages and/or B cells) incells isolated from presently or previously M. leprae-infectedindividuals. A M. leprae-infected individual may be afflicted with aform of leprosy (such as paucibacillary (PB), multibacillary (MB),lepromatous leprosy (LL), borderline lepromatous (BL), mid-borderline(BB), borderline tuberculoid (BT), or tuberculoid leprosy (Ti)) or maybe asymptomatic. Such individuals may be identified using methods knownto those of ordinary skill in the art. Individuals with leprosy may beidentified based on clinical findings associated with, for example, atleast one of the following: appearance of hypopigmented or reddishlesion with hypoesthesia, presence of acid fast bacilli in lymph nodesmears and compatible skin lesion histopathology. Asymptomaticindividuals are infected individuals who have no signs or symptoms ofthe disease. Such individuals can be identified, for example, based on apositive serological test and/or skin test.

The term “PBMC,” which refers to a preparation of nucleated cellsconsisting primarily of lymphocytes and monocytes that are present inperipheral blood, encompasses both mixtures of cells and preparations ofone or more purified cell types. PBMC may be isolated by methods knownto those in the art. For example, PBMC may be isolated by densitycentrifugation through, for example, Ficoll™ (Winthrop Laboratories, NewYork). Lymph node cultures may generally be prepared by immunizingBALB/c mice (e.g., in the rear foot pad) with M. leprae promastigotesemulsified in complete Freund's adjuvant. The draining lymph nodes maybe excised following immunization and T-cells may be purified in ananti-mouse Ig column to remove the B cells, followed by a passagethrough a Sephadex G10 column to remove the macrophages. Similarly,lymph node cells may be isolated from a human following biopsy orsurgical removal of a lymph node.

The ability of a fusion polypeptide of the disclosure to induce aresponse in PBMC or lymph node cell cultures may be evaluated, forexample, by contacting the cells with the polypeptide and measuring asuitable response. In general, the amount of polypeptide that issufficient for the evaluation of about 2×10⁵ cells ranges from about 10ng to about 100 ug or 100 ng to about 50 ug, and preferably is about 1ug, to 10 ug. The incubation of polypeptide (e.g., a fusion polypeptide)with cells is typically performed at 37° C. for about 1-3 days.Following incubation with polypeptide, the cells are assayed for anappropriate response. If the response is a proliferative response, anyof a variety of techniques well known to those of ordinary skill in theart may be employed. For example, the cells may be exposed to a pulse ofradioactive thymidine and the incorporation of label into cellular DNAmeasured. In general, a polypeptide that results in at least a threefold increase in proliferation above background (i.e., the proliferationobserved for cells cultured without polypeptide) is considered to beable to induce proliferation.

Alternatively, the response to be measured may be the secretion of oneor more cytokines (such as interferon-y (IFN-γ), interleukin-4 (IL-4),interleukin-12 (p70 and/or p40), interleukin-2 (IL-2) and/or tumornecrosis factor-a (TNF-α)) or the change in the level of mRNA encodingone or more specific cytokines. For example, the secretion ofinterferon-y, interleukin-2, tumor necrosis factor-a and/orinterleukin-12 is indicative of a Th1 response, which contributes to theprotective effect against M. leprae. Assays for any of the abovecytokines may generally be performed using methods known to those ofordinary skill in the art, such as an enzyme-linked immunosorbent assay(ELISA). Suitable antibodies for use in such assays may be obtained froma variety of sources such as Chemicon, Temucula, CA and PharMingen, SanDiego, CA, and may generally be used according to the manufacturer'sinstructions. The level of mRNA encoding one or more specific cytokinesmay be evaluated by, for example, amplification by polymerase chainreaction (PCR). In general, a polypeptide that is able to induce, in apreparation of about 1-3×10⁵ cells, the production of 30 pg/mL of IL-12,IL-4, IFN-γ, TNF-α or IL-12 p40, or 10 pg/mL of IL-12 p70, is consideredable to stimulate production of a cytokine.

Polynucleotide Compositions

The present disclosure also provides isolated polynucleotides,particularly those encoding the polypeptide combinations and/or fusionpolypeptides of the disclosure, as well as compositions comprising suchpolynucleotides. As used herein, the terms “DNA” and “polynucleotide”and “nucleic acid” refer to a DNA molecule that has been isolated freeof total genomic DNA of a particular species. Therefore, a DNA segmentencoding a polypeptide refers to a DNA segment that contains one or morecoding sequences yet is substantially isolated away from, or purifiedfree from, total genomic DNA of the species from which the DNA segmentis obtained. Included within the terms “DNA segment” and“polynucleotide” are DNA segments and smaller fragments of suchsegments, and also recombinant vectors, including, for example,plasmids, cosmids, phagemids, phage, viruses, and the like.

As will be understood by those skilled in the art, the polynucleotidesequences of this disclosure can include genomic sequences,extra-genomic and plasmid-encoded sequences and smaller engineered genesegments that express, or may be adapted to express, proteins, fusionpolypeptides, peptides and the like. Such segments may be naturallyisolated, recombinant, or modified synthetically by the hand of man.

As will be recognized by the skilled artisan, polynucleotides may besingle-stranded (coding or anti sense) or double-stranded, and may beDNA (genomic, cDNA or synthetic) or RNA molecules. Any polynucleotidemay be further modified to increase stability in vivo. Possiblemodifications include, but are not limited to, the addition of flankingsequences at the 5′ and/or 3′ ends; the use of phosphorothioate or 2′0-methyl rather than phosphodiesterase linkages in the backbone; and/orthe inclusion of nontraditional bases such as inosine, queosine andwybutosine, as well as acetyl- methyl-, thio- and other modified formsof adenine, cytidine, guanine, thymine and uridine. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide of the present disclosure, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a M. leprae antigen or a portion thereof) or maycomprise a variant, or a biological or antigenic functional equivalentof such a sequence. In particular embodiments, polynucleotides mayencode for two or more antigenic/immunogenic portions, fragments, orvariants derived from the M. leprae antigens described herein. In someembodiments, polynucleotides of the present disclosure comprise asequence encoding any of the immunogenic portions described herein. Insome embodiments, the polynucleotide comprises the sequence of SEQ IDNO: 1, 3, 5, 7, 9, or 11. Of course, portions of these sequences andvariant sequences sharing identity to these sequences may also beemployed (e.g., those having at least about any of 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% thereto).

Polynucleotide variants may contain one or more substitutions,additions, deletions and/or insertions, as further described below,preferably such that the immunogenicity of the encoded polypeptide isnot diminished, relative to the native protein. The effect on theimmunogenicity of the encoded polypeptide may generally be assessed asdescribed herein.

For example, in certain embodiments, variants of the disclosure includecysteine-modified polynucleotides in which the cysteine-encoding codonsare replaced with codons encoding other amino acids not capable offorming intrachain or interchain disulfide bonds. In more specificembodiments, some or all of the replacement codons encode serine becauseof the spatial similarity of the serine sidechain to the cysteinesidechain in the resulting polypeptide. In another specific embodiment,some or all of the replacement codons encode alanine. Illustrativemethods of replacing cysteine and other codons within a polynucleotideare well known (e.g., U.S. Pat. No. 4,816,566, the contents of which areincorporated herein by reference, and Proc Natl Acad Sci 97 (15): 8530,2000).

The term “variants” also encompasses homologous genes of xenogenicorigin.

In additional embodiments, isolated polynucleotides of the presentdisclosure comprise various lengths of contiguous stretches of sequenceidentical to or complementary to the sequence encoding M. lepraepolypeptides, such as those sequences disclosed herein. For example,polynucleotides are provided by this disclosure that comprise at leastabout 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 ormore contiguous nucleotides of two or more of the sequences disclosedherein as well as all intermediate lengths there between. It will bereadily understood that “intermediate lengths”, in this context, meansany length between the quoted values, such as 16, 17, 18, 19, etc.; 21,22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102,103, etc.; 150, 151, 152, 153, etc.; including all integers through200-500; 500-1,000, and the like.

The polynucleotides of the present disclosure, or fragments thereof,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a polynucleotidefragment of almost any length may be employed; with the total lengthpreferably being limited by the ease of preparation and use in theintended recombinant DNA protocol.

Moreover, it will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide as described herein. Someof these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent disclosure, for example polynucleotides that are optimized forhuman and/or primate codon selection. Further, alleles of the genescomprising the polynucleotide sequences provided herein are within thescope of the present disclosure. Alleles are endogenous genes that arealtered as a result of one or more mutations, such as deletions,additions and/or substitutions of nucleotides. The resulting mRNA andprotein may, but need not, have an altered structure or function.Alleles may be identified using standard techniques (such ashybridization, amplification and/or database sequence comparison).

M. leprae polynucleotides and fusions thereof may be prepared,manipulated and/or expressed using any of a variety of well establishedtechniques known and available in the art. In particular embodiments,fusions comprise two or more polynucleotide sequences encoding M. lepraepolypeptides.

For example, polynucleotide sequences or fragments thereof which encodepolypeptides of the disclosure, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of a polypeptide in appropriate host cells. Due to theinherent degeneracy of the genetic code, other DNA sequences that encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and express agiven polypeptide of the present disclosure.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce fusion polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

Moreover, the polynucleotide sequences of the present disclosure can beengineered using methods generally known in the art in order to alterfusion polypeptide encoding sequences for a variety of reasons,including but not limited to, alterations which modify the cloning,processing, expression and/or immunogenicity of the gene product.

In order to express a desired fusion polypeptide comprising two or moreantigenic/immunogenic fragments or portions of M. leprae polypeptides, anucleotide sequence encoding the fusion polypeptide, or a functionalequivalent, may be inserted into appropriate expression vector, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted coding sequence. Methods which are wellknown to those skilled in the art may be used to construct expressionvectors containing sequences encoding a polypeptide of interest andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook et al., Molecular Cloning, A Laboratory Manual(2001), and Ausubel et al., Current Protocols in Molecular Biology(January 2008, updated edition).

A variety of expression vector/host systems are known and may beutilized to contain and express polynucleotide sequences. These include,but are not limited to, microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast (such as Saccharomyces or Pichia) transformed with yeastexpression vectors; insect cell systems infected with virus expressionvectors (e.g., baculovirus); plant cell systems transformed with virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322plasmids); or animal cell systems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector-enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORTI plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the expressed polypeptide. Forexample, when large quantities are needed, vectors which direct highlevel expression of fusion proteins that are readily purified may beused. Such vectors include, but are not limited to, the multifunctionalE. coli cloning and expression vectors such as PBLUESCRIPT (Stratagene),in which the sequence encoding the polypeptide of interest may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of B-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke & Schuster, J. Biol. Chem.264:5503 5509 (1989)); and the like, pGEX Vectors (Promega, Madison,Wis.) may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al., Methods Enzymol. 153:516-544 (1987).

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, EMBO J. 6:307-311 (1987)).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi et EMBO J. 3:1671-1680 (1984);Broglie et al., Science 224:838-843 (1984); and Winter et al., ResultsProbl. Cell Differ. 17:85-105 (1991)). These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (see, e.g., Hobbs in McGraw Hill,Yearbook of Science and Technology, pp. 191-196 (1992)).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which the polypeptide of interest may be expressed (Engelhardet al., Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227 (1994)).

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of the presentdisclosure may be ligated into an adenovirus transcription/translationcomplex consisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad.Sci. U.S.A. 81:3655-3659 (1984)). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a fusion polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, et al., ResultsProbL Cell Differ. 20:125-162 (1994)).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed fusion protein in the desired fashion. Such modifications ofthe polypeptide include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. Post-translational processing which cleaves a “prepro” formof the protein may also be used to facilitate correct insertion, foldingand/or function. Different host cells such as CHO, HeLa, MDCK, HEK293,and W138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a fusion polynucleotide of the present disclosure may betransformed using expression vectors which may contain viral origins ofreplication and/or endogenous expression elements and a selectablemarker gene on the same or on a separate vector. Following theintroduction of the vector, cells may be allowed to grow for 1-2 days inan enriched media before they are switched to selective media. Thepurpose of the selectable marker is to confer resistance to selection,and its presence allows growth and recovery of cells which successfullyexpress the introduced sequences. Resistant clones of stably transformedcells may be proliferated using tissue culture techniques appropriate tothe cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler et al., Cell 11:223-232 (1977)) and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817-823 (1990)) geneswhich can be employed in tk- or aprt- cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler et al., Proc. Natl. Acad. Sci. U.S.A. 77:3567-70(1980)); npt, which confers resistance to the aminoglycosides, neomycinand G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); andals or pat, which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl.Acad. Sci. U.S.A. 85:8047-51 (1988)). The use of visible markers hasgained popularity with such markers as anthocyanins, B-glucuronidase andits substrate GUS, and luciferase and its substrate luciferin, beingwidely used not only to identify transformants, but also to quantify theamount of transient or stable protein expression attributable to aspecific vector system (Rhodes et al., Methods MoL Biol. 55:121-131(1995)).

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). These and otherassays are described, among other places, in Hampton et al., SerologicalMethods, a Laboratory Manual (1990) and Maddox et al., I Exp. Med.158:1211-1216 (1983).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide.

Alternatively, the sequences, or any portions thereof may be cloned intoa vector for the production of an mRNA probe. Such vectors are known inthe art, are commercially available, and may be used to synthesize RNAprobes in vitro by addition of an appropriate RNA polymerase such as T7,T3, or SP6 and labeled nucleotides. These procedures may be conductedusing a variety of commercially available kits. Suitable reportermolecules or labels, which may be used, include radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides of thedisclosure may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins. In addition to recombinant production methods, fusionpolypeptides of the disclosure, and fragments thereof, may be producedby direct peptide synthesis using solid-phase techniques (Merrifield, J.Am. Chem. Soc. 85:2149-2154 (1%3)). Protein synthesis may be performedusing manual techniques or by automation. Automated synthesis may beachieved, for example, using Applied Biosystems 431 A PeptideSynthesizer (Perkin Elmer). Alternatively, various fragments, forexample, immunogenic fragments from M. leprae polypeptides, may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

Pharmaceutical and Vaccine Compositions

In certain aspects, the polypeptides, antigens, polynucleotides,portions, variants, fusion polypeptides, etc., as described herein, areincorporated into pharmaceutical compositions or vaccines.Pharmaceutical compositions generally comprise one or more polypeptides,antigens, polynucleotides, portions, variants, fusion polypeptides,etc., as described herein, in combination with a physiologicallyacceptable carrier. Vaccines, also referred to as immunogeniccompositions, generally comprise one or more of the polypeptides,antigens, polynucleotides, portions, variants, fusion proteins, etc., asdescribed herein, in combination with an immunostimulant, such as anadjuvant. In particular embodiments, the compositions comprise fusionpolypeptides containing M. leprae antigens (or portions or variantsthereof) that are capable of providing protection against M. leprae. Insome embodiments, the compositions comprise fusion polypeptidescontaining M. leprae antigens (or portions or variants thereof) that arecapable of providing protection against a tuberculosis-causingMycobacterium.

An immunostimulant may be any substance that enhances or potentiates animmune response (antibody and/or cell-mediated) to an exogenous antigen.Examples of immunostimulants include adjuvants, biodegradablemicrospheres (e.g., polylactic galactide) and liposomes (into which thecompound is incorporated; see, e.g., Fullerton, U.S. Pat. No.4,235,877). Vaccine preparation is generally described in, for example.Powell & Newman, eds., Vaccine Design (the subunit and adjuvantapproach) (1995).

Any of a variety of immunostimulants may be employed in the vaccines ofthis disclosure. For example, an adjuvant may be included. Manyadjuvants contain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A (natural or synthetic), Bordatellapertussis or Mycobacterium species or Mycobacterium-derived proteins.Suitable adjuvants are commercially available as, for example, Freund'sIncomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2and derivatives thereof (GlaxoSmithKline Beecham, Philadelphia, Pa.);CWS, TDM, LeIF, aluminum salts such as aluminum hydroxide gel (alum) oraluminum phosphate; salts of calcium, iron or zinc; an insolublesuspension of acylated tyrosine; acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quit A. Cytokines, such asGM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

Certain embodiments of the present disclosure contemplate vaccine andpharmaceutical compositions that include one or more toll-like receptoragonists (TLR agonist). In more specific embodiments, for example, thecompositions of the disclosure include Toll-like receptor agonists, suchas TLR7 agonists and TLR7/8 agonists. In certain embodiments the TLRagonist is capable of delivering a biological signal by interacting withat least one TLR that is selected from TLR-2, TLR-3, TLR-4, TLR-5,TLR-6, TLR-7, TLR-8 and TLR-9.

Toll-like receptors (TLR) include cell surface transmembrane receptorsof the innate immune system that confer early-phase recognitioncapability to host cells for a variety of conserved microbial molecularstructures such as may be present in or on a large number of infectiouspathogens, (e.g., Armant et al., 2002 Genome Biol. 3(8): reviews3011.1-3011.6; Fearon et al., 1996 Science 272:50; Medzhitov et al.,1997 Curr. Opin. Immunol. 9:4; Luster 2002 Curr. Opin. Immunol. 14:129;Lien et al. 2003 Nat. Immunol. 4:1162; Medzhitov, 2001 Nat. Rev.Immunol. 1:135; Takeda et al., 2003 Ann Rev Immunol. 21:335; Takeda etal. 2005 Inf Immunol. 17:1; Kaisho et al., 2004 Microbes Infect. 6:1388;Datta et al., 2003 J. Immunol. 170:4102).

Induction of TLR-mediated signal transduction to potentiate theinitiation of immune responses via the innate immune system may beeffected by TLR agonists, which engage cell surface TLR or cytoplasmicTLR. For example, lipopolysaccharide (LPS) may be a TLR agonist throughTLR2 or TLR4 (Tsan et al., 2004 J. Leuk. Biol. 76:514; Tsan et al., 2004Am. J. Physiol. Cell Phsiol. 286: C739; Lin et al., 2005 Shock 24:206);poly(inosine-cytidine) (polyl:C) may be a TLR agonist through TLR3(Salem et al., 2006 Vaccine 24:5119); CpG sequences(oligodeoxynucleotides containing unmethylated cytosine-guanosine or“CpG” dinucleotide motifs, e.g., CpG 7909, Cooper et al., 2005 AIDS19:1473; CpG 10101 Bayes et al. Methods Find Exp Clin Pharnacol 27:193;Vollmer et al. Expert Opinion on Biological Therapy 5:673; Vollmer etal., 2004 Antimicrob. Agents Chemother. 48:2314; Deng et al., 2004 J.Immunol. 173:5148) may be TLR agonists through TLR9 (Andaloussi et a.,2006 Glia 54:526; Chen et al., 2006 J. Immunol. 177:2373);peptidoglycans may be TLR2 and/or TLR6 agonists (Soboll et al., 2006Biol. Reprod. 75:131; Nakao et al., 2005 J. Immunol. 174:1566); 3M003(4-amino-2-(ethoxymethyl)-a,a-dimethyl-617,8,9-tetrahydro-IH-imidazo[4,5]quinoline-1-ethanol hydrate, Mol. Wt. 318 Da from 3M Pharmaceuticals,St. Paul, MN, which is also a source of the related compounds 3M001 and3M002; Gorden et al., 2005 J. Immunol. 174:1259) may be a TLR7 agonist(Johansen 2005 Clin. Exp. Allerg. 35:1591) and/or a TLR8 agonist(Johansen 2005); flagellin may be a TLR5 agonist (Feuillet et al., 2006Proc. Nat. Acad. Sci. USA 103: 12487); and hepatitis C antigens may actas TLR agonists through TLR7 and/or TLR9 (Lee et al., 2006 Proc. Nat.Acad. Sci. USA 103:1828; Horsmans et al., 2005 Hepatol. 42:724). OtherTLR agonists are known (e.g., Schirmbeck et al., 2003 J. Immunol.171:5198) and may be used according to certain of the presentlydescribed embodiments.

For example, and by way of background (see, e.g., U.S. Pat. No.6,544,518) immunostimulatory oligonucleotides containing unmethylatedCpG dinucleotides (“CpG”) are known as being adjuvants when administeredby both systemic and mucosal routes (WO 96/02555, EP 468520, Davis etal., J. Immunol, 1998. 160(2): 870-876; McCluskie and Davis, J.Immunol., 1998, 161(9):4463-6). CpG is an abbreviation forcytosine-guanosine dinucleotide motifs present in DNA. The central roleof the CG motif in immunostimulation was elucidated by Krieg, Nature374, p546 1995. Detailed analysis has shown that the CG motif has to bein a certain sequence context, and that such sequences are common inbacterial DNA but are rare in vertebrate DNA. The immunostimulatorysequence is often: Purine. Purine, C, G, pyrimidine, pyrimidine: whereinthe dinucleotide CG motif is not methylated, but other unmethylated CpGsequences are known to be immunostimulatory and may be used in certainembodiments of the present disclosure. CpG when formulated intovaccines, may be administered in free solution together with freeantigen (WO 96/02555; McCluskie and Davis, supra) or covalentlyconjugated to an antigen (PCT Publication No. WO 98/16247), orformulated with a carrier such as aluminium hydroxide (e.g., Davis etal. supra, Brazolot-Millan et al., Proc.NatLAcad.Sci., USA, 1998,95(26), 15553-8).

Other illustrative oligonucleotides for use in compositions of thepresent disclosure will often contain two or more dinucleotide CpGmotifs separated by at least three, more preferably at least six or morenucleotides. The oligonucleotides of the present disclosure aretypically deoxynucleotides. In one embodiment the internucleotide in theoligonucleotide is phosphorodithioate, or more preferably aphosphorothioate bond, although phosphodiester and other intemucleotidebonds are within the scope of the disclosure including oligonucleotideswith mixed internucleotide linkages. Methods for producingphosphorothioate oligonucleotides or phosphorodithioate are described inU.S. Pat. Nos. 5,666,153, 5,278,302 and WO95/26204.

Other examples of oligonucleotides have sequences that are disclosed inthe following publications; for certain herein disclosed embodiments thesequences preferably contain phosphorothioate modified intemucleotidelinkages: CPG 7909: Cooper et al., “CPG 7909 adjuvant improves hepatitisB virus vaccine seroprotection in antiretroviral-treated HIV-infectedadults.” AIDS, 2005 Sep. 23; 19(14): 1473-9.

CpG 10101: Bayes et al., “Gateways to clinical trials.” Methods Find.Exp. Clin.

Pharmacol. 2005 April; 27(3): 193-219.

Vollmer J., “Progress in drug development of immunostimula-tory CpGoligodeoxynucleotide ligands for TLR9.” Expert Opinion on BiologicalTherapy. 2005 May; 5(5): 673-682.

Alternative CpG oligonucleotides may comprise variants of the preferredsequences described in the above-cited publications that differ in thatthey have inconsequential nucleotide sequence substitutions, insertions,deletions and/or additions thereto. The CpG oligonucleotides utilized incertain embodiments of the present disclosure may be synthesized by anymethod known in the art (e.g., EP 468520). Conveniently, sucholigonucleotides may be synthesized utilizing an automated synthesizer.The oligonucleotides are typically deoxynucleotides. In a preferredembodiment the intemucleotide bond in the oligonucleotide isphosphorodithioate, or more preferably phosphorothioate bond, althoughphosphodiesters are also within the scope of the presently contemplatedembodiments. Oligonucleotides comprising different internucleotidelinkages are also contemplated, e.g., mixed phosphorothioatephophodiesters. Other internucleotide bonds which stabilize theoligonucleotide may also be used.

In certain more specific embodiments the TLR agonist is selected fromlipopolysaccharide, peptidoglycan, polyl: C, CpG, 3M003, flagellin, M.leprae homolog of eukaryotic ribosomal elongation and initiation factor4a (LeIF) and at least one hepatitis C antigen.

Still other illustrative adjuvants include imiquimod, gardiquimod andresiquimod (all available from Invivogen), and related compounds, whichare known to act as TLR7/8 agonists. A compendium of adjuvants that maybe useful in vaccines is provided by Vogel et al., Pharm Biotechnol6:141 (1995), which is herein incorporated by reference.

Compositions of the disclosure may also employ adjuvant systems designedto induce an immune response predominantly of the Th1 type. High levelsof Th1-type cytokines (e.g., IFN-γ, TNF-a, IL-2 and IL-12) tend to favorthe induction of cell mediated immune responses to an administeredantigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4,IL-5, IL-6 and IL-10) tend to favor the induction of humoral immuneresponses. Following application of a vaccine as provided herein, apatient will support an immune response that includes Th1- and Th2-typeresponses. Within a preferred embodiment, in which a response ispredominantly of the Th1-type, the level of Th1-type cytokines willincrease to a greater extent than the level of Th2-type cytokines. Thelevels of these cytokines may be readily assessed using standard assays.For a review of the families of cytokines, see Mossman & Coffman, Ann.Rev. Immunol. 7:145-173 (1989).

Certain adjuvants for use in eliciting a predominantly Th1-type responseinclude, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL™), togetherwith an aluminum salt (U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034;and 4,912,094). CpG-containing oligonucleotides (in which the CpGdinucleotide is unmethylated) also induce a predominantly Th1 response.Such oligonucleotides are well known and are described, for example, inWO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008.200 and 5,856.462.Immunostimulatory DNA sequences are also described, for example, by Satoet al., Science 273:352 (1996). Another illustrative adjuvant comprisesa saponin, such as Quil A, or derivatives thereof, including QS21 andQS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.); Escin;Digitonin; or Gypsophila or Chenopodium quinoa saponins. Otherillustrative formulations include more than one saponin in the adjuvantcombinations of the present disclosure, for example combinations of atleast two of the following group comprising QS21, QS7, Quil A, 0-escin,or digitonin.

In a particular embodiment, the adjuvant system includes the combinationof a monophosphoryl lipid A and a saponin derivative, such as thecombination of QS21 and 3D-MPL™ adjuvant, as described in WO 94/00153,or a less reactogenic composition where the QS21 is quenched withcholesterol, as described in WO 96/33739. Other formulations comprise anoil-in-water emulsion and tocopherol. Another adjuvant formulationemploying QS21, 3D-MPL™ adjuvant and tocopherol in an oil-in-wateremulsion is described in WO 95/17210.

In certain preferred embodiments, the adjuvant used in the presentdisclosure is a glucopyranosyl lipid A (GLA) adjuvant, as described inU.S. Patent Application Publication No. 20080131466, the disclosure ofwhich is incorporated herein by reference in its entirety. In oneembodiment, the GLA adjuvant used in the context of the presentdisclosure has the following structure:

where: R¹, R³, R⁵ and R⁶ are C₁₁-C₂₀ alkyl, and R² and R⁴ are C₉-C₂₀alkyl.

In a more specific embodiment, the GLA has the formula set forth abovewherein R¹, R³, R⁵ and R⁶ are C₁₁₋₁₄ alkyl, and R² and R⁴ are C₁₂₋₁₅alkyl.

In a more specific embodiment, the GLA has the formula set forth abovewherein R¹, R³, R⁵ and R⁶ are C₁₁ alkyl; and R² and R⁴ are C₁₃ alkyl.

In a more specific embodiment, the GLA has the formula set forth abovewherein R¹, R³, R⁵ and R⁶ are C₁₁ alkyl; and R² and R⁴ are C₉ alkyl.

In certain embodiments, the adjuvant is a GLA adjuvant (e.g., synthetic)having the following structure:

In certain embodiments of the above GLA structure, R¹, R³, R⁵ and R⁶ areC₁₁-C₂₀ alkyl; and R² and R⁴ are C₉-C₂₀ alkyl. In certain embodiments,R¹, R³, R⁵ and R⁶ are C₁₁ alkyl; and R² and R⁴ are C₉ alkyl.

In certain embodiments, the adjuvant is a synthetic GLA adjuvant havingthe following structure:

In certain embodiments of the above GLA structure, R¹, R³, R⁵ and R⁶ areC₁₁-C₂₀ alkyl; and R² and R⁴ are C₉-C₂₀ alkyl. In certain embodiments.R¹, R³, R⁵ and R⁶ are C₁₁ alkyl; and R² and R⁴ are C₉ alkyl.

In certain embodiments, the adjuvant is a synthetic GLA adjuvant havingthe following structure:

In certain embodiments of the above GLA structure. R¹, R³, R⁵ and R⁶ areC₁₁-C₂₀ alkyl; and R² and R⁴ are C₁₁-C₂₀ alkyl. In certain embodiments.R¹, R³, R⁵ and R⁶ are C₁₁ alkyl; and R² and R⁴ are C₉ alkyl.

In certain embodiments, the adjuvant is a synthetic GLA adjuvant havingthe following structure:

In certain embodiments, the adjuvant is a synthetic GLA adjuvant havingthe following structure:

In certain embodiments, the adjuvant is a synthetic GLA adjuvant havingthe following structure:

In certain embodiments, the adjuvant is GLA-SE having the followingstructure:

In certain embodiments, the adjuvant is GLA-SE having the followingstructure:

The skilled artisan will understand that, in any of the embodimentsdescribed herein, the GLA adjuvant may be in a salt form, e.g., anammonium salt.

GLA-SE refers to a stable oil-in-water emulsion comprising GLAformulated in squalene oil and other excipients including, for example,dimyristoyl phosphatidyl choline (DPMC). In some preferred embodiments,20 ug/ml GLA is formulated in 4% squalene oil. Methods of making GLA-SEare known in the art, see for example, Misquith et al., Colloids andSurfaces B: Biointerfaces 113(2014) 312-319; Fox et al., Vaccine31(2013) 1633-1640, Van Hoeven et al., Nature Scientific Reports7:46426.

Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative as disclosed inWO 00/09159.

Other illustrative adjuvants include Montanide ISA 720 (Seppic, France),SAF (Chiron, Calif, United States), ISCOMS (CSL), MF-59 (Chiron), theSBAS series of adjuvants (e.g., SBAS-2, AS2′, AS2,″ SBAS-4, or SBAS6,available from SmithKline Beecham, Rixensart, Belgium), Detox, RC-529(Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide4-phosphates (AGPs), such as those described in pending U.S. patentapplication Ser. Nos. 08/853,826 and 09/074,720, the disclosures ofwhich are incorporated herein by reference in their entireties, andpolyoxyethylene ether adjuvants such as those described in WO99/52549A1.

The vaccine and pharmaceutical compositions of the disclosure may beformulated using any of a variety of well known procedures. In certainembodiments, the vaccine or pharmaceutical compositions are prepared asstable emulsions (e.g., oil-in-water emulsions) or as aqueous solutions.

Compositions of the disclosure may also, or alternatively, comprise Tcells specific for fusion polypeptide comprising immunogenic/antigenicportions or fragments of M. leprae antigens or variants thereof,described herein. Such cells may generally be prepared in vitro or exvivo, using standard procedures. For example, T cells may be isolatedfrom bone marrow, peripheral blood, or a fraction of bone marrow orperipheral blood of a patient. Alternatively, T cells may be derivedfrom related or unrelated humans, non-human mammals, cell lines orcultures.

T cells may be stimulated with a fusion polypeptide comprising M. lepraepolypeptides or immunogenic portions or variants thereof, polynucleotideencoding such a fusion polypeptide, and/or an antigen presenting cell(APC) that expresses such a fusion polypeptide. Such stimulation isperformed under conditions and for a time sufficient to permit thegeneration of T cells that are specific for the polypeptide. In certainembodiments, the polypeptide or polynucleotide is present within adelivery vehicle, such as a microsphere, to facilitate the generation ofspecific T cells.

T cells are considered to be specific for a fusion polypeptide of thedisclosure if the T cells specifically proliferate, secrete cytokines orkill target cells coated with the fusion polypeptide or expressing agene encoding the fusion polypeptide. T cell specificity may beevaluated using any of a variety of standard techniques. For example,within a chromium release assay or proliferation assay, a stimulationindex of more than two fold increase in lysis and/or proliferation,compared to negative controls, indicates T cell specificity. Such assaysmay be performed, for example, as described in Chen et al., Cancer Res.54:1065-1070 (1994)).

Alternatively, detection of the proliferation of T cells may beaccomplished by a variety of known techniques. For example, T cellproliferation can be detected by measuring an increased rate of DNAsynthesis (e.g., by pulse-labeling cultures of T cells with tritiatedthymidine and measuring the amount of tritiated thymidine incorporatedinto DNA). Contact with a polypeptide of the disclosure (100ng/ml-1001.1 g/ml, preferably 200 ng/ml-251.1 g/ml) for 3-7 days shouldresult in at least a two fold increase in proliferation of the T cells.Contact as described above for 2-3 hours should result in activation ofthe T cells, as measured using standard cytokine assays in which a twofold increase in the level of cytokine release (e.g., TNF or IFN-γ) isindicative of T cell activation (see Coligan et al., Current Protocolsin Immunology, vol. 1 (1998)). T cells that have been activated inresponse to a polypeptide, polynucleotide or polypeptide-expressing APCmay be CD4+ and/or CD8+. Protein-specific T cells may be expanded usingstandard techniques. Within preferred embodiments, the T cells arederived from a patient, a related donor or an unrelated donor, and areadministered to the patient following stimulation and expansion.

In the compositions of the disclosure, formulation ofpharmaceutically-acceptable excipients and carrier solutions iswell-known to those of skill in the art, as is the development ofsuitable dosing and treatment regimens for using the particularcompositions described herein in a variety of treatment regimens,including e.g., oral, parenteral, intravenous, intranasal, intradermal,subcutaneous and intramuscular administration and formulation.

In certain applications, the compositions disclosed herein may bedelivered via oral administration to a subject. As such, thesecompositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

In certain circumstances it will be desirable to deliver thecompositions disclosed herein parenterally, intravenously,intramuscularly, or even intraperitoneally as described, for example, inU.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each specificallyincorporated herein by reference in its entirety). Solutions of theactive compounds as free base or pharmacologically acceptable salts maybe prepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions may also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be facilitated by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion (see, e.g., Remington's PharmaceuticalSciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with thevarious other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxy groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective fortreatment of leprosy. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known to one ofordinary skill in the art. Except insofar as any conventional media oragent is incompatible with the active ingredient, its use in thetherapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood to one of ordinary skill in the art. Typically, suchcompositions are prepared as injectables, either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection can also be prepared. The preparation can alsobe emulsified.

In certain embodiments, the compositions of the present disclosure maybe delivered by intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering genes, polynucleotides, andpeptide compositions directly to the lungs via nasal aerosol sprays hasbeen described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (eachspecifically incorporated herein by reference in its entirety).Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S.Pat. No. 5,725,871, specifically incorporated herein by reference in itsentirety) are also well-known in the pharmaceutical arts. Likewise,transmucosal drug delivery in the form of a polytetrafluoroethylenesupport matrix is described in U.S. Pat. No. 5,780,045 (specificallyincorporated herein by reference in its entirety).

In certain embodiments, the delivery may occur by use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of compositions comprising a fusionpolypeptide as describe herein into suitable host cells. In particular,the compositions of the present disclosure may be formulated fordelivery either encapsulated in a lipid particle, a liposome, a vesicle,a nanosphere, a nanoparticle or the like. The formulation and use ofsuch delivery vehicles can be carried out using known and conventionaltechniques.

A pharmaceutical or immunogenic composition may, alternatively, containan immunostimulant and a nucleic acid molecule, e.g., a DNA or RNAmolecule encoding one or more of the polypeptides or fusion polypeptidesas described above, such that a desired polypeptide is generated insitu. In such compositions, the DNA encoding the fusion protein may bepresent within any of a variety of delivery systems known to those ofordinary skill in the art, including nucleic acid expression systems,bacteria and viral expression systems. Appropriate nucleic acidexpression systems contain the necessary DNA sequences for expression inthe patient (such as a suitable promoter and terminating signal).Bacterial delivery systems involve the administration of a bacterium(such as Bacillus-Calmette-Guerrin) that expresses an immunogenicportion of the polypeptide on its cell surface. In a particularembodiment, the DNA may be introduced using a viral expression system(e.g., vaccinia or other pox virus, retrovirus, or adenovirus), whichmay involve the use of a non-pathogenic (defective), replicationcompetent virus. Techniques for incorporating DNA into such expressionsystems are well known to those of ordinary skill in the art. The DNAmay also be “naked.” as described, for example, in Ulmer et al., Science259:1745-1749 (1993) and reviewed by Cohen, Science 259: 1691-1692(1993). The uptake of naked DNA may be increased by coating the DNA ontobiodegradable beads, which are efficiently transported into the cells.

The pharmaceutical compositions and vaccines of the disclosure may beused, in certain embodiments, to induce protective immunity against M.leprae in a patient, such as a human or an armadillo, to prevent leprosyor diminish its severity. The compositions and vaccines may also be usedto stimulate an immune response, which may be cellular and/or humoral,in a patient, for treating an individual already infected. In oneembodiment, for M. Leprae-infected patients, the immune responsesgenerated include a preferential Th1 immune response (i.e., a responsecharacterized by the production of the cytokines interleukin-1,interleukin-2, interleukin-12 and/or interferon-y, as well as tumornecrosis factor-a). In another embodiment, for uninfected patients, theimmune response involves production of interleukin-12 and/orinterleukin-2, or the stimulation of gamma delta T-cells. In eithercategory of patient, the response stimulated may include IL-12production. Such responses may also be elicited in biological samples ofPBMC or components thereof derived from M. leprae-infected or uninfectedindividuals. As noted above, assays for any of the above cytokines, aswell as other known cytokines, may generally be performed using methodsknown to those of ordinary skill in the art, such as an enzyme-linkedimmunosorbent assay (ELISA).

Appropriate doses and methods of fusion polypeptide administration forthese purposes can be readily determined by a skilled artisan usingavailable knowledge in the art and/or routine techniques. Routes andfrequency of administration, as well as dosage, for the above aspects ofthe present disclosure may vary from individual to individual and mayparallel those currently being used in immunization against otherinfections, including protozoan, viral and bacterial infections. Forexample, in one embodiment, between 1 and 12 doses of composition havinga fusion polypeptide, which comprises M. leprae polypeptides orimmunogenic/antigenic portions, fragments or variants thereof, areadministered over a 1-year period. Booster vaccinations may be givenperiodically thereafter as needed or desired. Of course, alternateprotocols may be appropriate for individual patients. In a particularembodiment, a suitable dose is an amount of fusion polypeptide or DNAencoding such a peptide that, when administered as described above, iscapable of eliciting an immune response in an immunized patientsufficient to protect the patient from leprosy caused by M. leprae. Ingeneral, the amount of fusion polypeptide present in a dose (or producedin situ by the DNA in a dose) ranges from about 100 ng to about 1 mg perkg of host, typically from about 101.1 g to about 100 ug. Suitable dosesizes will vary with the size of the patient, but will typically rangefrom about 0.1 mL to about 5 mL. In some aspects, from 1 ug to about 20ug per dose or from about 1 ug to about 10 ug per dose of a compositionof the present invention is administered to a subject in the methodsdescribed herein. If so desired, the composition can be, for example, inlyophilized form. In some aspects, the composition is administered incombination with an immunostimulant. The immunostimulant can be, forexample, any of the immunostimulants described herein. In some aspects,the immunostimulant is GLA having any one of the structures describedherein and is optionally formulated in an oil-in-water emulsion. In someaspects, the GLA is administered at a dose of from 2 ug to 20 ug perdose, or from about 1 ug to about 10 ug per dose or at about 5 ug perdose. The skilled artisan will appreciate that alternative dosageamounts are contemplated herein.

Methods of Stimulating an Immune Response

In another aspect, this disclosure provides methods for stimulating animmune response against M. leprae in a mammal including the step ofadministering to a mammal in need thereof a composition of the presentdisclosure. In some embodiments, the methods further include a step ofadministering to the mammal M. bovis BCG vaccine. In other embodimentsM. bovis BCG vaccine was previously administered to the mammal. Themethod may involve stimulating an immune response in various populationsof mammals, including, where the mammal has not been exposed to M.leprae, where the mammal has been exposed to M. leprae, where the mammalis a human healthy household contact of a human identified as beinginfected with M. leprae, where the mammal has been infected by M.leprae, and where the mammal exhibits signs or symptoms of infection byM. leprae. The compositions of the present disclosure can beadministered, for example, prophylactically, post-exposure but prior toclinical symptoms, or post-exposure and after exhibition of clinicalsymptoms. In some aspects, it will be unknown whether or not the mammalto be treated has been exposed to M. leprae but the mammal will havebeen in a leprosy endemic region or in contact with a mammal havingactive leprosy.

In another aspect, the disclosure provides methods for stimulating animmune response against a tuberculosis-causing mycobacterium in a mammalcomprising administering to a mammal in need thereof a composition ofthe disclosure.

Methods of Treatment

In another aspect, the disclosure provides methods for treating an M.leprae infection in a mammal, including the step of administering to amammal having an M. leprae infection a composition of the disclosure.The method may include multiple subsequent administrations of thecomposition.

Identifying mammals having an M. leprae infection may be carried withmethods known in the art. The World Health Organization (WHO) hasestablished diagnostic criteria as the presence of one or more of thefollowing key signs: appearance of hypopigmented or reddish lesion withhypoesthesia, presence of acid fast bacilli in lymph node smears andcompatible skin lesion histopathology. Once diagnosed, leprosy istreatable and patients are operationally defined into one of twocategories, paucibacillary (PB) or multibacillary (MB) for treatmentpurposes. The Ridley-Jopling scale characterizes five forms of leprosythrough the use of clinical, histopathological, and immunologicalmethods: lepromatous leprosy (LL), borderline lepromatous (BL),mid-borderline (BB), borderline tuberculoid (BT), and tuberculoidleprosy (TT). {Ridley D S et al., Int J Lepr Other Mycobact Dis 1966;34(3): 255-73; Scollard D M Int. J Lepr Other Mycobact Dis 2004; 72(2):166-8.} A pure neural leprosy presentation, which is PB, also exists. PBleprosy patients, encompassing TT and a number of BT forms, arecharacterized as having one or few skin lesions and granulomatousdermatopathology with a low or absent bacterial index (BI). At theextreme PB pole, TT patients demonstrate a specific cell-mediatedimmunity against M. leprae and have an absent, or low, BI. Control ofbacterial growth by PB patients indicates that these individuals mount astrong, but not necessarily curative, immune response against M. leprae.

In some embodiments, the methods further include a step of administeringto the mammal one or more chemotherapeutic agents. A “chemotherapeutic”.“chemotherapeutic agents” or “chemotherapy regime” is a drug orcombination of drugs used to treat or in the treatment thereof ofpatients infected or exposed to M. leprae and includes, but is notlimited to, amikacin, aminosalicylic acid, capreomycin, clofazimine,cycloserine, dapsone, ethambutol, ethionamide, gatifloxacin, isoniazid(INH), kanamycin, linezolid, minocycline, pyrazinamide, rifamycins(i.e., rifampin, rifampicin, rifapentine and rifabutin), streptomycin,ofloxacin, ciprofloxacin, clarithromycin, azithromycin, PA824, andfluoroquinolones and other derivatives analogs or biosimilars in theart.

In some embodiments, the mammal is first administered one or morechemotherapeutic agents over a period of time and subsequentlyadministered the composition. In other embodiments, the mammal is firstadministered the composition and subsequently administered one or morechemotherapeutic agents over a period of time. In other embodiments,administration of the one or more chemotherapeutic agents and thecomposition is concurrent.

In some embodiments, the method includes a step of administering to themammal M. bovis BCG vaccine. In other embodiments, M. bovis BCG vaccinewas previously administered to the mammal.

The method may be practiced on various groups of mammals. In someembodiments, the mammal does not exhibit signs or symptoms of infectionby M. leprae. In some embodiments, the mammal has indeterminate ortuberculoid presentation. In some embodiments, the mammal haspaucibacillary leprosy. In some embodiments, the mammal hasmultibacillary leprosy. In some embodiments, the mammal has lepromatousleprosy. In some embodiments, the mammal has borderline lepromatousleprosy. In some embodiments, the mammal has mid-borderline leprosy. Insome embodiments, the mammal has borderline tuberculoid leprosy. In someembodiments, the mammal has tuberculoid leprosy. In some embodiments,the mammal is infected with a multidrug resistant M. leprae. In someembodiments, the mammal is a human.

In another aspect, a method for reducing the time course of chemotherapyagainst an M. leprae infection is provided. The time course ofchemotherapy is shortened, for example, to no more than about 3 months,about 5 months, or about 7 months.

A “chemotherapeutic”, “chemotherapeutic agents” or “chemotherapy regime”is a drug or combination of drugs used to treat or in the treatmentthereof of patients infected or exposed to M. leprae and includes, butis not limited to, amikacin, aminosalicylic acid, capreomycin,clofazimine, cycloserine, dapsone, ethambutol, ethionamide,gatifloxacin, isoniazid (INH), kanamycin, linezolid, minocycline,pyrazinamide, rifamycins (i.e., rifampin, rifampicin, rifapentine andrifabutin), streptomycin, ofloxacin, ciprofloxacin, clarithromycin,azithromycin, PA824, and fluoroquinolones and other derivatives analogsor biosimilars in the art.

In another aspect, the disclosure provides methods for preventing an M.leprae infection in a mammal, or preventing onset of clinical symptomsin a mammal that has been exposed to M. leprae or has been diagnosedwith M. leprae but does not yet exhibit symptoms thereof, including thestep of administering to a mammal having an M. leprae infection acomposition of the disclosure. The method may include multiplesubsequent administrations of the composition.

It will also be understood that the methods of treatment of the presentdisclosure may include the administration of the compositions of thedisclosure either alone or in conjunction with other agents and, assuch, the therapeutic vaccine may be one of a plurality of treatmentcomponents as part of a broader therapeutic treatment regime.Accordingly, the methods of the present disclosure advantageouslyimprove the efficacy of a chemotherapy treatment regime for thetreatment of M. leprae infection.

In another aspect, the present disclosure provides kits for treatment ofan M. leprae infection including a composition of the disclosure.

Diagnostic Compositions, Methods and Kits

In another aspect, this disclosure provides compounds and methods fordetecting leprosy in individuals and in blood supplies. In particularembodiments, the individual is a mammal. In more particular embodiments,the mammal is a human or armadillo.

For example, the fusion polypeptides, polypeptides, and antigens of thepresent disclosure can be used as effective diagnostic reagents fordetecting and/or monitoring M. leprae infection in a patient. Forexample, the compositions, fusion polypeptides, and polynucleotides ofthe disclosure may be used in in vitro and in vivo assays for detectinghumoral antibodies or cell-mediated immunity against M. leprae fordiagnosis of infection, monitoring of disease progression ortest-of-cure evaluation. In particular embodiments, the fusionpolypeptides and polynucleotides are useful diagnostic reagents forserodiagnosis and whole blood assay in patients having leprosy or inindividuals exposed to M. leprae.

In one aspect, the diagnostic methods and kits preferably employ acomposition or fusion polypeptide as described herein, repeats ofpolypeptide fragments, or multimeric polypeptide fragments, includingantigenic/immunogenic fragments. In another more specific aspect, fusionpolypeptides of the present disclosure may comprise two or more M.leprae antigen fragments. In a more particular embodiment, anillustrative fusion polypeptide comprises the amino acid sequence setforth in SEQ ID NO: 12. In another embodiment, the diagnostic methodsand kits preferably employ a fusion polypeptide comprising at least 1,at least 2, at least 3, or at least 4 immunogenic/antigenic portions orfragments of M. leprae polypeptides, variants or the like, optionally incombination with one or more other M. leprae antigens or non-M. lepraesequences, as described herein or obtainable in the art.

The antigens or polypeptides may be used in essentially any assay formatdesired, e.g., as individual antigens assayed separately, as multipleantigens assays simultaneously (e.g., a fusion polypeptide), as antigensimmobilized on a solid support such as an array, or the like.

In one embodiment, there are provided diagnostic kits for detecting M.leprae infection in a biological sample, comprising (a) a polypeptide ora fusion polypeptide described herein or variants thereof as describedherein, and (b) a detection reagent.

In another embodiment, there are provided diagnostic kits for detectingM. leprae infection in a biological sample, comprising (a) antibodies orantigen binding fragments thereof that are specific for a polypeptide ora fusion polypeptides described herein or variants thereof as describedherein, and (b) a detection reagent.

In another embodiment, methods are provided for detecting the presenceof M. leprae infection in a biological sample, comprising (a) contactinga biological sample with a polypeptide or a fusion polypeptide describedherein or variants thereof described herein; and (b) detecting in thebiological sample the presence of antibodies that bind to the fusionpolypeptide.

In another embodiment, methods are provided for detecting the presenceof M. leprae infection in a biological sample, comprising (a) contactinga biological sample with at least 2 monoclonal antibodies that bind to apolypeptide or a polypeptide described herein or variants thereofdescribed herein; and (b) detecting in the biological sample thepresence of M. leprae proteins that bind to the monoclonal antibody.

One of ordinary skill in the art would recognize that the methods andkits described herein may be used to detect all types of leprosy,depending on the particular combination of immunogenic portions of M.leprae antigens present in the fusion polypeptide.

There are a variety of assay formats known to those of ordinary skill inthe art for using a fusion polypeptide to detect antibodies in a sample.See, e.g., Harlow and Lane, Antibodies. A Laboratory Manual, Cold SpringHarbor Laboratory Press, 1988, which is incorporated herein byreference. In one embodiment, the assay involves the use of fusionpolypeptide immobilized on a solid support to bind to and remove theantibody from the sample. The bound antibody may then be detected usinga detection reagent that binds to the antibody/peptide complex andcontains a detectable reporter group. Suitable detection reagents arewell known and include, for example, antibodies that bind to theantibody/polypeptide complex and free polypeptide labeled with areporter group (e.g., in a semi-competitive assay). Suitable reportergroups are also well known and include, for example, fluorescent labels,enzyme labels, radioisotopes, chemiluminescent labels,electrochemiluminescent labels, bioluminescent labels, polymers, polymerparticles, metal particles, haptens, and dyes. Alternatively, acompetitive assay may be utilized, in which an antibody that binds to afusion polypeptide of the present disclosure labeled with a reportergroup and allowed to bind to the immobilized fusion polypeptide afterincubation of the fusion polypeptide with the sample. The extent towhich components of the sample inhibit the binding of the labeledantibody to the fusion polypeptide is indicative of the reactivity ofthe sample with the immobilized fusion polypeptide.

The solid support may be any material known to those of ordinary skillin the art to which the fusion polypeptide may be attached. For example,the support may be a test well in a microtiter plate or a nitrocelluloseor other suitable membrane. Alternatively, the support may be a bead ordisc, such as glass, fiberglass, latex or a plastic material such aspolystyrene or polyvinylchloride. The support may also be a magneticparticle or a fiber optic sensor, such as those disclosed, for example,in U.S. Pat. No. 5,359,681.

The fusion polypeptide may be bound to the solid support using a varietyof techniques known to those in the art, which are amply described inthe patent and scientific literature. In the context of the presentdisclosure, the term “bound” refers to both non-covalent association,such as adsorption, and covalent attachment (which may be a directlinkage between the antigen and functional groups on the support or maybe a linkage by way of a cross-linking agent). Binding by adsorption toa well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the polypeptide, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and 1 day. In general, contacting a well of a plastic microtiterplate (such as polystyrene or polyvinylchloride) with an amount offusion polypeptide ranging from about 10 ng to about 1 pg, andpreferably about 100 ng, is sufficient to bind an adequate amount ofantigen. Nitrocellulose will bind approximately 100 pg of protein per 3cm.

Covalent attachment of fusion polypeptide to a solid support maygenerally be achieved by first reacting the support with a bifunctionalreagent that will react with both the support and a functional group,such as a hydroxyl or amino group, on the fusion polypeptide. Forexample, the fusion polypeptide may be bound to a support having anappropriate polymer coating using benzoquinone or by condensation of analdehyde group on the support with an amine and an active hydrogen onthe polypeptide (see, e.g., Pierce Immunotechnology Catalog and Handbook(1991) at A12-A13).

In certain embodiments, the assay is an enzyme linked immunosorbentassay (ELISA). This assay may be performed by first contacting a fusionpolypeptide of the present disclosure that has been immobilized on asolid support, commonly the well of a microtiter plate, with the sample,such that antibodies to the M. leprae antigens of the fusion polypeptidewithin the sample are allowed to bind to the immobilized fusionpolypeptide. Unbound sample is then removed from the immobilized fusionpolypeptide and a detection reagent capable of binding to theimmobilized antibody-polypeptide complex is added. The amount ofdetection reagent that remains bound to the solid support is thendetermined using a method appropriate for the specific detectionreagent.

Once the fusion polypeptide is immobilized on the support, the remainingprotein binding sites on the support are typically blocked. Any suitableblocking agent known to those of ordinary skill in the art, such asbovine serum albumin (BSA) or Tween 20™ (Sigma Chemical Co., St. Louis,Mo.) may be employed. The immobilized polypeptide is then incubated withthe sample, and antibody (if present in the sample) is allowed to bindto the antigen. The sample may be diluted with a suitable diluent, suchas phosphate-buffered saline (PBS) prior to incubation. In general, anappropriate contact time (i.e., incubation time) is that period of timethat is sufficient to permit detection of the presence of antibodywithin a M. leprae-infected sample. Preferably, the contact time issufficient to achieve a level of binding that is at least 95% of thatachieved at equilibrium between bound and unbound antibody. Those ofordinary skill in the art will recognize that the time necessary toachieve equilibrium may be readily determined by assaying the level ofbinding that occurs over a period of time. At room temperature, anincubation time of about 30 minutes is generally sufficient.

Unbound sample may then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% Tween 20™. Detectionreagent may then be added to the solid support. An appropriate detectionreagent is any compound that binds to the immobilizedantibody-polypeptide complex and that can be detected by any of avariety of means known to those in the art. Preferably, the detectionreagent contains a binding agent (such as, for example, Protein A.Protein G, immunoglobulin, lectin or free antigen) conjugated to areporter group. Preferred reporter groups include enzymes (such ashorseradish peroxidase), substrates, cofactors, inhibitors, dyes,radionuclides, luminescent groups, fluorescent groups, colloidal goldand biotin. The conjugation of binding agent to reporter group may beachieved using standard methods known to those of ordinary skill in theart. Common binding agents may also be purchased conjugated to a varietyof reporter groups from many sources (e.g., Zymed Laboratories, SanFrancisco, Calif, and Pierce, Rockford, Ill.).

The detection reagent is then incubated with the immobilized antibodypolypeptide complex for an amount of time sufficient to detect the boundantibody. An appropriate amount of time may generally be determined fromthe manufacturer's instructions or by assaying the level of binding thatoccurs over a period of time. Unbound detection reagent is then removedand bound detection reagent is detected using the reporter group. Themethod employed for detecting the reporter group depends upon the natureof the reporter group. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of anti-M. leprae antibodies in thesample, the signal detected from the reporter group that remains boundto the solid support is generally compared to a signal that correspondsto a predetermined cut-off value. In one embodiment, the cut-off valueis preferably the average mean signal obtained when the immobilizedpolypeptide is incubated with samples from an uninfected patient. Ingeneral, a sample generating a signal that is three standard deviationsabove the predetermined cut-off value is considered positive (i.e.,reactive with the polypeptide). In an alternate embodiment, the cut-offvalue is determined using a Receiver Operator Curve, according to themethod of Sackett et al., Clinical Epidemiology: A Basic Science forClinical Medicine, p. 106-7 (Little Brown and Co., 1985). Briefly, inthis embodiment, the cut-off value may be determined from a plot ofpairs of true positive rates (i.e., sensitivity) and false positiverates (100%-specificity) that correspond to each possible cut-off valuefor the diagnostic test result. The cut-off value on the plot that isthe closest to the upper lefthand corner (i.e., the value that enclosesthe largest area) is the most accurate cut-off value, and a samplegenerating a signal that is higher than the cut-off value determined bythis method may be considered positive. Alternatively, the cut-off valuemay be shifted to the left along the plot, to minimize the falsepositive rate, or to the right, to minimize the false negative rate.

In other embodiments, an assay is performed in a flow-through assayformat, wherein the antigen is immobilized on a membrane such asnitrocellulose. In the flow-through test, antibodies within the samplebind to the immobilized polypeptide as the sample passes through themembrane. A detection reagent (e.g., protein A-colloidal gold) thenbinds to the antibody-polypeptide complex as the solution containing thedetection reagent flows through the membrane. The detection of bounddetection reagent may then be performed as described above.

In other embodiments, an assay if performed in a strip test format, alsoknown as a lateral flow format. Here, one end of the membrane to whichpolypeptide is bound is immersed in a solution containing the sample.The sample migrates along the membrane through a region containingdetection reagent and to the area of immobilized fusion polypeptide.Concentration of detection reagent at the fusion polypeptide indicatesthe presence of M. leprae antibodies in the sample. Typically, theconcentration of detection reagent at that site generates a pattern,such as a line, that can be read visually. The absence of such a patternindicates a negative result. In general, the amount of fusionpolypeptide immobilized on the membrane is selected to generate avisually discernible pattern when the biological sample contains a levelof antibodies that would be sufficient to generate a positive signal inan ELISA, as discussed above. Preferably, the amount of fusionpolypeptide immobilized on the membrane ranges from about 25 ng to about1 fag, and more preferably from about 50 ng to about 500 ng. Such testscan typically be performed with a very small amount (e.g., one drop) ofpatient serum or blood. Lateral flow tests can operate as eithercompetitive or sandwich assays.

In still other embodiments, a fusion polypeptide of the disclosure isadapted for use in a dual path platform (DPP) assay. Such assays aredescribed, for example, in U.S. Pat. No. 7,189,522, the contents ofwhich are incorporated herein by reference.

Of course, numerous other assay protocols exist that are suitable foruse with the fusion polypeptides of the present disclosure. It will beunderstood that the above descriptions are intended to be exemplaryonly.

The assays discussed above may be used, in certain aspects of thedisclosure, to specifically detect visceral leprosy. In this aspect,antibodies in the sample may be detected using a fusion polypeptide ofthe present disclosure, e.g., comprising an amino acid sequence ofantigenic/immunogenic fragments or epitopes of M. leprae antigens.Preferably, the M. leprae antigens are immobilized by adsorption to asolid support such as a well of a microtiter plate or a membrane, asdescribed above, in roughly similar amounts such that the total amountof fusion polypeptide in contact with the support ranges from about 10ng to about 100 pg. The remainder of the steps in the assay maygenerally be performed as described above. It will be readily apparentto those of ordinary skill in the art that, by combining polypeptidesdescribed herein with other polypeptides that can detect cutaneous andmucosal leprosy, the polypeptides disclosed herein may be used inmethods that detect all types of leprosy.

In another aspect of this disclosure, immobilized fusion polypeptidesmay be used to purify antibodies that bind thereto. Such antibodies maybe prepared by any of a variety of techniques known to those of ordinaryskill in the art. See, e.g., Harlow and Land, Antibodies. A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1988. In one suchtechnique, an immunogen comprising a fusion polypeptide of the presentdisclosure is initially injected into any of a wide variety of mammals(e.g., mice, rats, rabbits, sheep and goats). In this step, thepolypeptide may serve as the immunogen without modification.Alternatively, particularly for relatively short polypeptides, asuperior immune response may be elicited if the polypeptide is joined toa carrier protein, such as bovine serum albumin or keyhole limpethemocyanin. The immunogen is injected into the animal host, preferablyaccording to a predetermined schedule incorporating one or more boosterimmunizations, and the animals are bled periodically. Polyclonalantibodies specific for the polypeptide may then be purified from suchantisera by, for example, affinity chromatography using the polypeptidecoupled to a suitable solid support.

Monoclonal antibodies specific for the antigenic fusion polypeptide ofinterest may be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. A preferred selectiontechnique uses HAT (hypoxanthine, aminopterin, thymidine) selection.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and tested for bindingactivity against the polypeptide. Hybridomas having high reactivity andspecificity are preferred.

Monoclonal antibodies may be isolated from the supematants of growinghybridoma colonies. In this process, various techniques may be employedto enhance the yield, such as injection of the hybridoma cell line intothe peritoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. One or more polypeptides may be used inthe purification process in, for example, an affinity chromatographystep.

Monospecific antibodies that bind to a fusion polypeptide comprising twoor more immunogenic portions of M. leprae antigens may be used, forexample, to detect M. leprae infection in a biological sample using oneof a variety of immunoassays, which may be direct or competitive.Briefly, in one direct assay format, a monospecific antibody may beimmobilized on a solid support (as described above) and contacted withthe sample to be tested. After removal of the unbound sample, a secondmonospecific antibody, which has been labeled with a reporter group, maybe added and used to detect bound antigen. In an exemplary competitiveassay, the sample may be combined with the monoclonal or polyclonalantibody, which has been labeled with a suitable reporter group. Themixture of sample and antibody may then be combined with polypeptideantigen immobilized on a suitable solid support. Antibody that has notbound to an antigen in the sample is allowed to bind to the immobilizedantigen and the remainder of the sample and antibody is removed. Thelevel of antibody bound to the solid support is inversely related to thelevel of antigen in the sample. Thus, a lower level of antibody bound tothe solid support indicates the presence of M. leprae in the sample.Other formats for using monospecific antibodies to detect M. leprae in asample will be apparent to those of ordinary skill in the art, and theabove formats are provided solely for exemplary purposes.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “apolypeptide” optionally includes two or more polypeptides, and the like.

It is understood that aspect and embodiments of the disclosure describedherein include “comprising,” “consisting,” and “consisting essentiallyof aspects and embodiments.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

EXAMPLES Example 1

Antigen Recognition in Leprosy Patient Samples.

Subjects and samples. Recently diagnosed and previously untreatedleprosy patients and endemic controls (EC) were recruited at Centra deReferencia em Diagnostico e Terapeutica and Hospital Anuar Auad,Goiania, Goias State, Brazil. Leprosy patients were categorized aspaucibacillary (PB) by clinical, bacilloscopic and histologicalobservations (bacterial index, skin lesions, nerve involvement andhistopathology) carried out by qualified personnel. Blood was obtainedfrom tuberculosis patients (Mycobacterium tuberculosis sputum-positive,HIV-negative individuals with clinically confirmed pulmonarytuberculosis) who were undergoing treatment. EC were healthy individualswho had never had tuberculosis, had no history of leprosy in the family,and were living in the leprosy endemic area. All donors had previouslybeen immunized with BCG and all blood samples were obtained afterinformed consent and after local ethics committee approval.

Determining reactivity by 24 hour whole blood assay (WBA). WBA wereperformed with venous undiluted heparinized whole blood (Greiner).Within 2 hours of collection, blood was added to each well of a 24-wellplate (450 μl/well: Sigma, St. Louis, MO) and incubated with antigens at37° C., 5% CO2. For each assay, stimulations were conducted with 10μg/ml of recombinant protein and 1 μg/ml PHA (Sigma). After 24 hours,plasma was collected and stored at −20° C. IFNγ content within theplasma was determined by ELISA, used according to the manufacturer'sinstructions (QuantiFERON CMI Cellestis, Camegie, Australia). Thedetection limit of the test was 0.05 IU/ml. For data interpretation, apositive result was assigned as a concentration above an arbitrarycut-off point of 0.5 IU/ml. Spontaneous IFNγ secretion was observed inWBA only for some TB patients, and in those cases, was subtracted toprovide antigen-induced values.

Antigen recognition by leprosy-affected individuals. Replication anddissemination of M. leprae is limited in PB leprosy patients and mostHHC, suggesting the potent cellular immune response they develop isassociated with limited or localized disease. Antigens that arerecognized by PB patients or HHC are potentially targets of an effectiveimmune response against M. leprae. To further the antigen selectionscheme, the analyses was expanded to include ethnically andgeographically distinct populations (Table 1). While each of theindividual antigens ML2028, ML2055 and ML2380 were recognized by over50% PB patients and HHC, the combination of these antigens pushed thetheoretical recognition to over 80%.

Table 1. Percent responders above 50 pg/ml IFNγ in WBA. Whole blood fromPB and HHC was cultured for 24 hours in the presence of antigen and IFNγcontent in the plasma was measured by ELISA. Data were generated withcohorts from Brazil and the Philippines.

TABLE 1 Percent responders above 50 pg/mL IFNγ in WBA #Posi- #Nega- %Posi- tive tive tive Total Re- Re- Re- Antigens # sponders sponderssponders observed ML2028 129 77 52 59.7 ML2025 130 90 40 69.2 ML2380 6845 23 66.2 theoretical ML2028 + ML2055 70 51 19 72.9 ML2028 + ML2380 3226 6 81.3 ML2055 + ML2380 32 27 5 84.4 ML2028 + 32 27 5 84.4 ML2055 +ML2380

Mice and immunizations. Wild type C57BL/6 (B6) mice were purchased fromCharles River Laboratories (Wilmington, MA). Mice were immunized withrecombinant protein formulated with saline, stable emulsion (SE), orglucopyranosyl lipid adjuvant (GLA)-SE, to provide a final proteinconcentration of 10 μg antigen and 20 μg GLA-SE. Mice were immunized upto 3 times by subcutaneous (s.c) injection of 0.1 ml volume at the baseof the tail at 2 week intervals. Mice were maintained in specificpathogen-free conditions and all procedures were approved by thepertinent institutional animal care and use committees.

Immunization with Select Antigens Reduces M. Leprae Infection.

Determination of bacterial burden. To assess M. leprae growth, live M.leprae bacilli (Thai-53 strain) were purified from the footpads of nu/numice at National Institute of Infectious Diseases. Mice were inoculatedwith 1×104 bacilli by s.c. injection into each foot pad. Foot pads wereharvested 12 months later and the bacilli were enumerated by directmicroscopic counting of acid-fast bacilli according to the method ofShepard and McRae or by RT-PCR of the M. leprae specific repetitiveelement (RLEP).

Immunization with select antigens reduces M. leprae infection. Toinvestigate if immunization with the recognized antigens could limit M.leprae infection, mice were immunized with single antigens, orcombinations of antigens, before infection with M. leprae. Immunizationsignificantly decreased bacterial numbers (FIGS. 1A and 1B;p-values<0.05). These data indicate that immunization with the selectedantigens elicits protective responses in mice and warrant inclusionwithin a defined sub-unit vaccine against leprosy.

Generation of ML89 Fusion Protein A single 89kD fusion protein,designated ML89, was generated from the ML2028, ML2055 and ML2380antigens, with the addition of ML2531 to stabilize expression.

Immune recognition of chimeric fusion protein ML89.

Antibody responses. Mouse sera were prepared following collection ofretro-orbital blood into microtainer serum collection tubes (VWRInternational, West Chester, PA) followed by centrifugation at 1200 rpmfor 5 minutes. Each serum was then analyzed by antibody capture ELISA.Briefly, ELISA plates (Nunc, Rochester, NY) were coated with 1 μg/mlrecombinant antigen in 0.1 M bicarbonate buffer and blocked with 1%BSA-PBS. Then, in consecutive order and following washes inPBS/Tween®20, serially diluted serum samples, anti-mouse IgG, IgG1 orIgG2c-HRP (all Southern Biotech, Birmingham, AL) and ABTS-H2O2(Kirkegaard and Perry Laboratories, Gaithersburg, MD) were added to theplates. Plates were analyzed at 405 nm (ELX808, Bio-Tek Instruments Inc,Winooski, VT). Endpoint titer was determined as the last dilution torender a positive response, determined as 2 times the mean opticaldensity of the replicates derived from sera from unimmunized mice inPrism software (GraphPad Software, La Jolla, CA).

Antigen stimulation and cytokine responses. Single cell suspensions wereprepared by disrupting spleens between frosted slides. Red blood cellswere removed by lysis in 1.66% NH₄Cl solution, then mononuclear cellsenumerated by Via Count assay with a PCA system (Guava Technologies,Hayward, CA). Single cell suspensions were cultured at 2×105 cells perwell in duplicate in a 96-well plate (Corning Incorporated, Corning, NY)in RPMI-1640 supplemented with 5% heat-inactivated FCS and 50,000 Unitspenicillin/streptomycin (Invitrogen). Cells were cultured in thepresence of 10 μg/ml antigen for 72-96 hours, after which culturesupematants were harvested and cytokine content assessed. Cytokineconcentrations within culture supematants were determined by singlecytokine ELISA or multiple cytokine luminex assays. ELIS A kits fordetermination of mouse IFNγ. IL-5, IL-13 and TNF-α were performedaccording to manufacturer's instructions (eBioscience, San Diego, CA)and optical density was determined using an ELx808 plate reader.

Immune recognition of chimeric fusion protein ML89. Combining multipleantigens into a single fusion protein is now commonly used to provide amore consistent production process whilst also increasing the proportionof the population that is likely to respond. We therefore created asingle 89kD fusion protein, designated ML89, consisting of the ML2028,ML2055 and ML2380 antigens, with the addition of ML2531 to stabilizeexpression. When mice were immunized with the ML89 they raisedantibodies against each individual component (FIGS. 2B-2D), indicatingthat antigenicity was retained. Given that the M. bovis BCG vaccine isroutinely used in leprosy-affected regions, we also examined theinterplay of ML89 and BCG vaccination and determined if prior BCGimmunization led to any interactions upon ML89 immunization. Mice wereeither primed with BCG or not, then immunized with ML89/GLA-SE.Subsequent analyses of the IFNγ recall response to ML89 indicatedsimilar responses in both immunization strategies (FIG. 3A).Furthermore, mice immunized with ML89 also responded to lysate of BCGand, most importantly, to crude M. leprae antigens (FIGS. 3B and 3D).These data indicate that recognition of ML89 raises responses thatrecognize M. leprae that are not adversely affected by prior BCGimmunization.

Immunization with ML89/GLA-SE reduces M. leprae burdens.

Determination of bacterial burden. To assess M. leprae growth, live M.leprae bacilli (Thai-53 strain) were purified from the footpads of nu/numice at National Institute of Infectious Diseases. Mice were inoculatedwith 1×104 bacilli by s.c. injection into each foot pad. Foot pads wereharvested 12 months later and the bacilli were enumerated by directmicroscopic counting of acid-fast bacilli according to the method ofShepard and McRae or by RT-PCR of the M. leprae specific repetitiveelement (RLEP).

Immunization with ML89/GLA-SE reduces M. leprae burdens. We hypothesizedthat immunization with the ML89 antigen would limit bacterial growth,and evaluated the ability of the ML89/GLA-SE vaccine candidate toprotect against experimental M. leprae infection using the BALB/c mousefootpad model. Immunized mice were infected with M. leprae in thefootpad and bacilli numbers assessed months later. The vaccine decreasedbacterial numbers by 85% when compared with mice injected with GLA-SEadjuvant alone (FIG. 4A; p-values<0.05). Immunization with ML89/GLA-SEelicited protection equivalent to the mixture of its individualcomponents, and could provide protection when injected 2 or 3 times(FIG. 4B; p-values<0.05). Taken together, the data indicated that thedefined subunit ML89/GLA-SE vaccine induces responses that control of M.leprae infection.

Immunization with ML89/GLA-SE reduced lymphadenopathy induced by M.leprae infection. Mice were injected s.c. with ML89/GLA-SE at biweeklyintervals, for a total of 3 immunizations. One month after the lastimmunization mice were infected with 1×106 M. leprae in each ear, andDLN cell numbers determined 16 weeks later. Results are shown as meanand SE (n=5 per group). Student's t-test was used to calculate p-valuesbetween each group.

ML89-specific T cells reduced M. leprae viability during experimentalinfection. Mice were injected s.c. with ML89/GLA-SE at biweeklyintervals, for a total of 3 immunizations. One month after the lastimmunization T cells were purified from the spleens of immunized miceand transferred by i.v. injection into athymic recipient mice. Recipientmice were infected with 1×104 M. leprae in each foot, and bacillinumbers and viability determined 1 month later. Results are shown asmean and SE. Mann-Whitney test was used to calculate p-values betweeneach group: n=6 per group.

Immunization with ML89/GLA-SE delays motor nerve function impairment

The manifestation of leprosy in nine-banded armadillo (Dasypusnovemcinctus), the only other natural host of M. leprae, is strikinglysimilar to humans. Most significantly, armadillos develop extensivenerve involvement during experimental M. leprae infection and canexhibit many classic clinical signs such as foot ulcers, skin lesionsand even blindness. Armadillos are an abundant source of leproticneurologic fibers and they have already provided some important insightsinto the demyelinating neuropathy involved in leprosy. Markedinflammation can be observed on histopathological inspection of infectedarmadillo nerves and a functional deficit can be demonstrated inleprotic nerves using electrophysiology. Importantly, among the uniqueattributes of experimental infection in armadillos are a controlled andknown infection status, and functional recapitulation of leprosy as seenin humans but with a compressed time until disease emergence.

Immunization with ML89/GLA-SE delays motor nerve function impairment.Given that the hallmark of leprosy is nerve damage, the vaccine wasevaluated in nine-banded armadillos that develop the nerve involvementand functional perturbations seen in humans. To mimic a situation thatmay commonly arise in leprosy hyper-endemic regions, namely asymptomaticM. leprae infection, armadillos were infected prior to immunization thenmonitored for motor nerve conduction abnormality. Untreated armadillosbegan to show nerve conduction deficits as early as 4 months afterinoculation, and all armadillos had had at least some measurable deficitby 12 months (FIG. 5A). Many animals that exhibited conduction deficitsone month demonstrated a return to normal measurements the next. Toaccount for these fluctuations, an animal demonstrating 3 consecutivemonths with abnormal readings was defined as exhibiting a sustaineddeficit. The variable nature of M. leprae infection in these outbredanimals became apparent using this parameter, with sustained nerveconduction deficits occurring 6-22 months after infection and 2 of 12(17%) infected armadillos not actually demonstrating persistentalterations (FIG. 5B). Interestingly. BCG immunization of alreadyinfected animals led to precipitation of nerve damage. While onset ofconduction deficits in BCG vaccinated armadillos occurred at the sametime as control untreated animals (FIG. 5 a ), sustained conductiondeficits were more rapidly observed in BCG vaccinated armadillos thancontrol untreated animals (FIG. 5 b ). The extent of the disseminationwas significant enough that 27% (3 of 11) of the BCG immunizedarmadillos had to be removed from the study. Sustained conductiondeficits were also more rapidly observed in BCG vaccinated armadillosthan control untreated animals. In stark contrast, LEP-F1/GLA-SEimmunization delayed the onset of motor nerve conduction abnormalityamong animals already incubating leprosy (FIG. 5B). It is highlypertinent that LEP-F1/GLA-SE immunization, at a minimum, appears to besafe and induces no further neurological injury in armadillos.

TIME OF GROUP ID SACRIFICE COMMENT BCG 11I203 12 months 11I302 12 months11I1903 10 months 12M41 19 months ID93 11J201 32 months Not duedissemination 11J301 23 months 11J901 13 months 12O68 23 months

Statistics. For human data, the Mann Whitney U test was applied forcomparison between two groups. The non-parametric Kruskal-Wallisanalysis of variance test was used to compare the IFNγ levels among allgroups. The p-values for mouse studies resulting in normally distributeddata including 2 groups were determined using the Student's t-test.Where more than 2 groups were compared, p-values were attained by ANOVAanalyses. Data were log-transformed for non-normal data sets prior toanalysis. Statistics were generated using MS Excel (MicrosoftCorporation, Redmond, WA) or Prism software (GraphPad Software, Inc., LaJolla, CA). Statistical significance was considered as p-values were<0.05.

DISCUSSION

Despite the positive impact that WHO-MDT has had on the globalprevalence of leprosy, there are many indications that further effortsare required to prevent the re-emergence of leprosy and continue effortstoward eradication. Targeting vaccination to at-risk populations,amongst which many individuals may already be infected with M. leprae,appears a tenable long lasting strategy. Many countries re-immunizeleprosy patients and their close contacts with the Mycobacterium bovisBCG vaccine developed against tuberculosis. Immunization with BCG doesafford some protection, although meta-analyses of clinical trialsestimated its ability to prevent leprosy to be modest (26% and 41%,respectively) (Setia et al., Lancet Infect. Dt. 2006:6(3): 162-170;Merle et al. Expert Review of Vaccines. 2010; 9(2):209-22.)

The persistence of leprosy in regions with good BCG coverage indicatesthat additional strategies are required.

Although M. leprae is killed by MDT, neurological injury continues tooccur in patients and can be exacerbated during inflammatory reactionalepisodes. Some clinicians/researchers fear that immunization to boostinflammatory T cell responses will induce nerve-damaging reversalreactions. Live attenuated or killed mycobacteria vaccines havegenerally been well tolerated in patients and the incidence of reactionshas not been dramatically altered versus unvaccinated groups, while morerapid bacterial clearance has occurred and has been accompanied bydistinct signs of clinical improvement. Anecdotal reports, and nowclinical evidence, indicate that BCG immunization may howeverprecipitate the onset of PB disease in some individuals, withspeculation that infected but asymptomatic M. leprae-infectedindividuals are at greatest risk. To date, however, the effect ofvaccination on M. leprae-associated neuropathy has not been investigatedin a controlled system. The data demonstrated that BCG vaccinationprecipitates nerve damage in M. leprae-infected armadillos, supportingthat hypothesis that infected individuals are at risk of diseaseprecipitation if vaccinated with BCG.

Thus, it was surprising that the data indicated that LEP-F1/GLA-SEimmunization was safe but also delayed nerve damage in animals infectedwith high doses of M. leprae.

As would be recognized by the skilled artisan, these and other changescan be made to the embodiments of the disclosure in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled.

Example 2

GLP Repeated Dose Toxicity Study In Rabbits

A study to determine the potential toxicity of LEP-F1+GLA-SE in NewZealand White rabbits when given every 14 days via IM injection for 6weeks as well as to determine if delayed toxicity and/or recoveryoccurred after a 4 week recovery period was performed. Systemic exposurewas evaluated by anti-LEP-F1 antibody analysis. Forty animals weredivided into two groups and received either saline or LEP-F1 (20ug)+GLA-SE (20 ug). The animals were dosed by IM Injection on Days 1,15, 29 and 43. None of the findings were considered to be highlytoxicologically significant and all had resolved by the end of therecovery period.

Example 3

Phase I Open Label Antigen Dose-Escalation Clinical Trial to Evaluatethe Safety, Tolerability, and Immunogenicity of LEP-F1+GLA-SE in HealthyAdult Subjects

To evaluate the safety and tolerability of 2 ug LEP-F1+5 ug GLA-SE and10 ug LEP-F1+5 ug GLA-SE following IM administration on study days 0,28, and 56 and to assess the immunogenicity of 2 ug LEP-F1+5 ug GLA-SEand 10 ug LEP-F1+5 ug GLA-SE by evaluating T cell responses to LEP-F1 atspecified time points, a Phase I clinical trial will be performed. Theproposed clinical trial is a first-in-man trial to establish an initialsafety profile in mycobacterially naive healthy adults. The evaluationof vaccine-induced immunity will be based on the development ofcirculating antibody and T cell responses directed against the LEP-F1antigen. Primary response will be assessed at Day 63. Responses atbaseline and Day 35 will also be assessed. Each participant will be onstudy for 14 months. Serum will be collected on days 0, 35, and 63.These samples will be used to determine by IgG ELISA whether subjectshave antibody responses to the LEP-F1 antigen at each of these timepoints. Measured antibody responses to LEP-F11 will be reported asnormalized titers. Cellular immune response analysis of selected Th1 andTh2 cytokines specific to LEP-F1 will be assayed on days 0, 35, and 63by whole blood assay. Cytokine concentrations will be quantified byELISA or multiplex bead array.

It is anticipated that adverse events will be generally mild, transientand typical of immunizations given by the IM. route. It is expected thatsubjects receiving LEP-F1+GLA-SE will have robust levels of antigenspecific IgG antibodies and will display antigen-specific CD4+ T cellresponses.

SEQUENCES SEQ ID NO: 1 Polynucleotide Encoding ML2028 AntigenATGATTGACGTGAGCGGGAAGATCCGAGCCTGGGGGCGCTGGCTTTTGGTGGGTGCAGCTGCGACTCTGCCGAGCCTAATCAGCCTTGCTGGCGGAGCGGCGACCGCAAGCGCGTTCTCACGACCAGGCCTACCCGTCGAGTACCTACAGGTGCCGTCGGAGGCGATGGGGCGCAGCACAAGGTGCAGTTTCAAAACGGCGGAAACGGCTCTCCGGCGGTGTATCTGCTGGATGGTTTGCGTGCGCAGGACGACTATAACGGCTGGGACATCAACACCTCCGCATTCGAGTGGTACTATCAGTCGGGACTCTCGGTCGTGATGCCGGTCGGTGGGCAATCCAGCTTCTACAGCGACTGGTACAGCCCAGCGTGCGGCAAGGCAGGTTGCACGACCTACAAGTGGGAAACATTCCTTACTAGCGAGCTGCCTAAATGGCTATCCGCCAATAGGAGTGTCAAATCCACCGGCAGCGCCGTGGTCGGCCTCTCGATGGCCGGTTCCTCGGCCCTAATACTGGCAGCTTATCACCCCGATCAGTTCATCTATGCTGGCTCGTTGTCGGCGCTGATGGACTOCTCCCAGGGGATAGAACCCCAGCTAATCGGCTTGGCGATGGGTGATGCTGGTGGCTACAAGGCCGCGGACATGTGGGGACCACCAAATGACCCGGCCTGGCAACGAAACGACCCCATTCTGCAGGCTGGGAAGCTGGTCGCCAACAACACCCACCTATGGGTTTACTGTGGTAACGGCACACCGTCAGAGTTGGGTGGAACCAACGTACCCGCGGAATTCCTGGAGAACTTCGTGCACGGCAGCAACCTAAAGTTCCAGGACGCCTACAACGGTGCTGGTGGCCACAACGCTGTGTTCAACCTCAATGCCGACGGAACGCACAGCTGGGAGTACTGGGGAGCCCAGCTCAACGCCATGAAGCCCGACCTACAGAACACCTTGATGGCTGTACCCCGCAGCGGTSEQ ID NO: 2 Amino Acid Sequence of ML2028 Antigen from Mycobacterium leprae(diacylglycerol acyltransferase; NCBI Reference Sequence: WP_010908679.1)MIDVSGKIRAWGRWLLVGAAATLPSLISLAGGAATASAFSRPGLPVEYLQVPSEAMGRSIKVQFQNGGNGSPAVYLLDGLRAQDDYNGWDINTSAFEWYYQSGLSVVMPVGGQSSFYSDWYSPACGKAGCTTYKWETFLTSELPKWLSANRSVKSTGSAVVGLSMAGSSALILAAYHPDQFIYAGSLSALMDSSQGIEPQLIGLAMGDAGGYKAADMWGPPNDPAWQRNDPILQAGKLVANNTHLWVYCGNGTPSELGGTNVPAEFLENFVHGSNLKFQDAYNGAGGHNAVFNLNADGTHSWEYWGAQLNAMKPDLQNTLMAVPRSGSEQ ID NO: 3 Polynucleotide Encoding ML2028₃₉₋₃₂₇TTCTCACGACCAGGCCTACCCGTCGAGTACCTACAGGTGCCGTCGGAGGCGATGGGGCGCAGCATCAAGGTGCAGTTTCAAAACGGCGGAAACGGCTCTCCGGCGGTGTATCTGCTGGATGGTTTGCGTGCGCAGGACGACTATAACGGCTGGGACATCAACACCTCCGCATTCGAGTGGTACTATCAGTCGGGACTCTCGGTCGTGATGCCGGTCGGTGGGCAATCCAGCTTCTACAGCGACTGGTACAGCCCAGCGTGCGGCAAGGCAGGTTGCACGACCTACAAGTGGGAAACATTCCTTACTAGCGAGCTGCCTAAATGGCTATCCGCCAATAGGAGTGTCAAATCCACCGGCAGCGCCGTGGTCGGCCTCTCGATGGCCGGTTCCTCGGCCCTAATACTGGCAGCTTATCACCCCGATCAGTTCATCTATGCTGGCTCGTTGTCGGCGCTGATGGACTCCTCCCAGGGGATAGAACCCCAGCTAATCGGCTTGGCGATGGGTGATGCTGGTGGCTACAAGGCCGCGGACATGTGGGGACCACCAAATGACCCGGCCTGGCAACGAAACGACCCCATTCTGCAGGCTGGGAAGCTGGTCGCCAACAACACCCACCTATGGGTTTACTGTGGTAACGGCACACCGTCAGAGTTGGGTGGAACCAACGTACCCGCGGAATTCCTGGAGAACTTCGTGCACGGCAGCAACCTAAAGTTCCAGGACGCCTACAACGGTGCTGGTGGCCACAACGCTGTGTTCAACCTCAATGCCGACGGAACGCACAGCTGGGAGTACTGGGGAGCCCAGCTCAACGCCATGAAGCCCGACCTACAGAACACCTTGATGGCTGTACCCCGCAGCGGTSEQ ID NO: 4 Amino Acid Sequence of Residues 39-327 of ML2028 antigen(ML2028₃₉₋₃₂₇)FSRPGLPVEYLQVPSEAMGRSIKVQFQNGGNGSPAVYLLDGLRAQDDYNGWDINTSAFEWYYQSGLSVVMPVGGQSSFYSDWYSPACGKAGCTTYKWETFLTSELPKWLSANRSVKSTGSAVVGLSMAGSSALILAAYHPDQFIYAGSLSALMDSSQGIEPQLIGLAMGDAGGYKAADMWGPPNDPAWQRNDPILQAGKLVANNTHLWVYCGNGTPSELGGTNVPAEFLENFVHGSNLKFQDAYNGAGGHNAVFNLNADGTHSWEYWGAQLNAMKPDLQNTLMAVPRSG SEQ ID NO: 5 Polynucleotide Encoding ML2055 AntigenATGAATCAGGTTGACCTGGACTCGACACATCGCAAAGGATTGTGGGCGATACTGGCGATTGCCGTGGTGGCCAGCGCCAGTGCCTTTACGATGCCGTTGCCTGCGGCCGCCAACGCCGATCCCGCGCCCCTGCCGCCATCGACGGCTACGGCAGCTCCCTCACCTGCGCAGGAGATCATTACACCCCTTCCAGGCGCCCCTGTCTCGTCCGAAGCCCAACCGGGTGATCCCAATGCGCCGTCGCTCGATCCGAATGCACCATACCCACTTGCAGTCGATCCCAACGCCGGCCGAATCACCAACGCTGTCGGTGGATTTAGCTTCGTCCTTCCTGCCGGTTGGGTGGAGTCAGAGGCTTCACATCTTGACTACGGTTCGGTGCTGCTCAGCAAAGCCATCGAGCAGCCGCCCGTGCTTGGTCAGCCGACGGTGGTCGCTACCGACACCCGTATAGTGCTCGGCCGGCTGGACCAAAAGCTCTACGCCAGTGCCGAAGCCGACAACATTAAGGCCGCGGTCCGACTGGGCTCGGATATGGGTGAGTTCTACCTGCCATACCCCGGTACGCGGATCAACCAAGAAACCATTCCGCTCCACGCCAACGGGATAGCTGGAAGCGCCTCCTACTACGAGGTCAAATTCAGCGATCCCAATAAGCCAATTGGCCAAATATGTACGAGCGTAGTCGGCTCGCCAGCGGCGAGTACCCCTGACGTGGGGCCCTCGCAGCGTTGGTTTGTGGTATGGCTCGGAACCTCGAATAACCCGGTGGACAAGGGCGCAGCCAAAGAGCTGGCTGAGTCTATCCGGTCAGAGATGGCTCCGATCCCGGCGTCGGTTTCCGCTCCGGCACCTGTTGGASEQ ID NO: 6 Amino Acid Sequence of ML2055 Antigen from Mycobacterium leprae(alanine and proline-rich secreted protein Apa; NCBI Reference Sequence:WP_010908692.1)MNQVDLDSTHRKGLWAILAIAVVASASAFTMPLPAAANADPAPLPPSTATAAPSPAQEIITPLPGAPVSSEAQPGDPNAPSLDPNAPYPLAVDPNAGRITNAVGGFSFVLPAGWVESEASHLDYGSVLLSKAIEQPPVLGQPTVVATDTRIVLGRLDQKLYASAEADNIKAAVRLGSDMGEFYLPYPGTRINQETIPLHANGIAGSASYYEVKFSDPNKPIGQICTSVVGSPAASTPDVGPSQRWFVVWLGTSNNPVDKGAAKELAESIRSEMAPIPASVSAPAPVG SEQ ID NO: 7 Polynucleotide Encoding ML2380 AntigenATGTCTCGGCTGAGCACCAGCCTATGTAAAGGTGCTGTTTTTCTCGTTTTCGGTATCATTCCTGTGGCATTTCCGACGACCGCCGTTGCCGATGGTTCCACGGAGGATTTTCCGATCCCCCGCAGGCAAATCGCCACCACCTGTGATGCAGAGCAGTATTTGGCGGCCGTCAGGGATACCAGCCCGATCTACTACCAGCGGTACATGATCGATATGCACAACAAGCCGACTGACATCCAGCAGGCCGCGGTCAATCGTATCCATTGGTTCTATTCCTTGAGCCCCACCGACCGTAGGCAGTATTCCGAGGACACCGCTACAAACGTCTACTACGAGCAGATGGCCACGCATTGGGGAAACTGGGCGAAGATTTTCTTCAATAACAAGGGCGTTGTCGCCAAAGCCACCGAGGTTTGCAACCAGTACCAGGCCGGAGACATGTCGGTGTGGAACTGGCCGSEQ ID NO: 8 Amino Acid Sequence of ML2380 Antigen from Mycobacterium leprae(hypothetical protein; NCBI Reference Sequence: WP_010908863.1)MSRLSTSLCKGAVFLVFGIIPVAFPTTAVADGSTEDFPIPRRQIATTCDAEQYLAAVRDTSPIYYQRYMIDMHNKPTDIQQAAVNRIHWFYSLSPTDRRQYSEDTATNVYYEQMATHWGNWAKIFFNNKGVVAKATEVCNQYQAGDMSVWNWP SEQ ID NO: 9 Polynucleotide Encoding ML2531 AntigenATGACACAGATTATGTACAACTACCCGGCAATGTTGGACCACGCCGGGAATATGTCAGCCTGCGCCGGCGCTTTGCAGGGGGGGGCATCGACATCGCTGCCGAGCAAGCTGCGTTGCAAGCTTGCTGGGGGGGCGATACTGGGATTAGTTATCAGGCCTGGCAGGTGCAGTGGAACCAGGCCACGGAAGAGATGGTGCGTGCCTACCATGCAATGGCCAACACTCACCAAAACAACACTTTGGCTATGCTCACCCGCGACCAAGCTGAAGCCGCCAAATGGGGCGGCSEQ ID NO: 10 Amino Acid Sequence of ML2531 Antigen from Mycobacterium leprae(ESAT-6-like protein EsxR; NCBI Reference Sequence: WP_010908945.1)MTQIMYNYPAMLDHAGNMSACAGALQGVGIDIAAEQAALQACWGGDTGISYQAWQVQWNQATEEMVRAYHAMANTHQNNTLAMLTRDQAEAAKWGGSEQ ID NO: 11 Polynucleotide Encoding the LEP-F1 Fusion PolypeptideATGACACAGATTATGTACAACTACCCGGCAATGTTGGACCACGCCGGGAATATGTCAGCCTGCGCCGGCGCTTTGCAGGGGGTGGGCATCGACATCGCTGCCGAGCAAGCTGCGTTGCAAGCTTGCTGGGGGGGCGATACTGGGATTAGTTATCAGGCCTGGCAGGTGCAGTGGAACCAGGCCACGGAAGAGATGGTGCGTGCCTACCATGCAATGGCCAACACTCACCAAAACAACACTTTGGCTATGCTCACCCGCGACCAAGCTGAAGCCGCCAAATGGGGCGGCGGATCCATGTCTCGGCTGAGCACCAGCCTATGTAAAGGTGCTGTTTTTCTCGTTTTCGGTATCATTCCTGTGGCATTTCCGACGACCGCCGTTGCCGATGGTTCCACGGAGGATTTTCCGATCCCCCGCAGGCAAATCGCCACCACCTGTGATGCAGAGCAGTATTTGGCGGCCGTCAGGGATACCAGCCCGATCTACTACCAGCGGTACATGATCGATATGCACAACAAGCCGACTGACATCCAGCAGGCCGCGGTCAATCGTATCCATTGGTTCTATTCCTTGAGCCCCACCGACCGTAGGCAGTATTCCGAGGACACCGCTACAAACGTCTACTACGAGCAGATGGCCACGCATTGGGGAAACTGGGCGAAGATTTTCTTCAATAACAAGGGCGTTGTCGCCAAAGCCACCGAGGTTTGCAACCAGTACCAGGCCGGAGACATGTCGGTGTGGAACTGGCCGGAGCTCATGAATCAGGTTGACCTGGACTCGACACATCGCAAAGGATTGTGGGCGATACTGGCGATTGCCGTGGTGGCCAGCGCCAGTGCCTTTACGATGCCGTTGCCTGCGGCCGCCAACGCCGATCCCGCGCCCCTGCCGCCATCGACGGCTACGGCAGCTCCCTCACCTGCGCAGGAGATCATTACACCCCTTCCAGGCGCCCCTGTCTCGTCCGAAGCCCAACCGGGTGATCCCAATGCGCCGTCGCTCGATCCGAATGCACCATACCCACTTGCAGTCGATCCCAACGCCGGCCGAATCACCAACGCTGTCGGTGGATTTAGCTTCGTCCTTCCTGCCGGTTGGGTGGAGTCAGAGGCTTCACATCTTGACTACGGTTCGGTGCTGCTCAGCAAAGCCATCGAGCAGCCGCCCGTGCTTGGTCAGCCGACGGTGGTCGCTACCGACACCCGTATAGTGCTCGGCCGGCTGGACCAAAAGCTCTACGCCAGTGCCGAAGCCGACAACATTAAGGCCGCGGTCCGACTGGGCTCGGATATGGGTGAGTTCTACCTGCCATACCCCGGTACGCGGATCAACCAAGAAACCATTCCGCTCCACGCCAACGGGATAGCTGGAAGCGCCTCCTACTACGAGGTCAAATTCAGCGATCCCAATAAGCCAATTGGCCAAATATGTACGAGCGTAGTCGGCTCGCCAGCGGCGAGTACCCCTGACGTGGGGCCCTCGCAGCGTTGGTTTGTGGTATGGCTCGGAACCTCGAATAACCCGGTGGACAAGGGCGCAGCCAAAGAGCTGGCTGAGTCTATCCGGTCAGAGATGGCTCCGATCCCGGCGTCGGTTTCCGCTCCGGCACCTGTTGGAGTCGACTTCTCACGACCAGGCCTACCCGTCGAGTACCTACAGGTGCCGTCGGAGGCGATGGGGCGCAGCATCAAGGTGCAGTTTCAAAACGGCGGAAACGGCTCTCCGGCGGTGTATCTGCTGGATGGTTTGCGTGCGCAGGACGACTATAACGGCTGGGACATCAACACCTCCGCATTCGAGTGGTACTATCAGTCGGGACTCTCGGTCGTGATGCCGGTCGGTGGGCAATCCAGCTTCTACAGCGACTGGTACAGCCCAGCGTGCGGCAAGGCAGGTTGCACGACCTACAAGTGGGAAACATTCCTTACTAGCGAGCTGCCTAAATGGCTATCCGCCAATAGGAGTGTCAAATCCACCGGCAGCGCCGTGGTCGGCCTCTCGATGGCCGGTTCCTCGGCCCTAATACTGGCAGCTTATCACCCCGATCAGTTCATCTATGCTGGCTCGTTGTCGGCGCTGATGGACTCCTCCCAGGGGATAGAACCCCAGCTAATCGGCTTGGCGATGGGTGATGCTGGTGGCTACAAGGCCGCGGACATGTGGGGACCACCAAATGACCCGGCCTGGCAACGAAACGACCCCATTCTGCAGGCTGGGAAGCTGGTCGCCAACAACACCCACCTATGGGTTTACTGTGGTAACGGCACACCGTCAGAGTTGGGTGGAACCAACGTACCCGCGGAATTCCTGGAGAACTTCGTGCACGGCAGCAACCTAAAGTTCCAGGACGCCTACAACGGTGCTGGTGGCCACAACGCTGTGTTCAACCTCAATGCCGACGGAACGCACAGCTGGGAGTACTGGGGAGCCCAGCTCAACGCCATGAAGCCCGACCTACAGAACACCTTGATGGCTGTACCCCGCAGCGGTSEQ ID NO: 12 Amino Acid Sequence of the LEP-F1 Fusion PolypeptideMTQIMYNYPAMLDHAGNMSACAGALQGVGIDIAAEQAALQACWGGDTGISYQAWQVQWNQATEEMVRAYHAMANTHQNNTLAMLTRDQAEAAKWGGGSMSRLSTSLCKGAVFLVFGIIPVAFPTTAVADGSTEDFPIPRRQIATTCDAEQYLAAVRDTSPIYYQRYMIDMHNKPTDIQQAAVNRIHWFYSL SPTDRRQYSEDTATNVYYEQMATHWGNWAKIFFNNKGVVAKATEVCNQYQAGDMSVWNWPELMNQVDLDSTHRKGLWAILAIAVVASASAFTMPLPAAANADPAPLPPSTATAAPSPAQEIITPLPGAPVSSEAQPGDPNAPSLDPNAPYPLAVDPNAGRITNAVGGFSFVLPAGWVESEASHLDYGSVLLSKAIEQPPVLGQPTVVATDTRIVLGRLDQKLYASAEADNIKAAVRLGSDMGEFYLPYPGTRINQETIPLHANGIAGSASYYEVKFSDPNKPIGQICTSVVGSPAASTPDVGPSQRWFVVWLGTSNNPVDKGAAKELAESIRSEMAPIPASVSAPAPVGVDFSRPGLPVEYLQVPSEAMGRSIKVQFQNGGNGSPAVYLLDGLRAQDDYNGWDINTSAFEWYYQSGLSVVMPVGGQSSFYSDWYSPACGKAGCTTYKWETFLTSELPKWLSANRSVKSTGSAVVGLSMAGSSALILAAYHPDQFIYAGSLSALMDSSQGIEPQLIGLAMGDAGGYKAADMWGPPNDPAWQRNDPILQAGKLVANNTHLWVYCGNGTPSELGGTNVPAEFLENFVHGSNLKFQDAYNGAGGHNAVFNLNADGTHSWEYWGAQLNAMKPDLQNTLMAVPRSG

TABLE 2 Results of alignment of ML2028 amino acid sequence with otherspecies NAME Cover Identity Accession diacylglycerol acyltransferase100% 94% WP_045843560.1 [Mycobacterium lepromatosis] diacylglycerolacyltransferase/ 100% 88% WP_047314133.1 mycolyltransferase Ag85A[Mycobacterium haemophilum] diacylglycerol acyltransferase/ 100% 84%WP_068052084.1 mycolyltransferase Ag85A [Mycobacterium sp. E342]diacylglycerol acyltransferase/ 100% 84% WP_067276075.1mycolyltransferase Ag85A [Mycobacterium scrofulaceum] diacylglycerolacyltransferase/ 100% 83% WP_068078824.1 mycolyltransferase Ag85A[Mycobacterium sp. E1747 diacylglycerol acyltransferase 100% 84%WP_046185518.1 [Mycobacterium nebraskense] diacylglycerolacyltransferase/  96% 85% WP_068048723.1 mycolyltransferase Ag85A[Mycobacterium sp. E2733] MULTISPECIES: diacylglycerol  96% 85%WP_067924124.1 acyltransferase/mycolytransferase Ag85A [Mycobacterium]Diacylglycerol acyltransferase/ 100% 83% Q50397.1 mycolyltransferaseAg85B [Mycobacterium scrofulaceum] diacylglycerol acyltransferase/  96%85% WP_066954325.1 mycolyltransferase Ag85A [Mycobacterium sp. 852002-53434_SCH5985345] diacylglycerol acyltransferase/  98% 83%WP_068023768.1 mycolyltransferase Ag85A [Mycobacterium szulgai]diacylglycerol acyltransferase/ 100% 83% WP_067099382.1mycolyltransferase Ag85A [Mycobacterium sp. 852002- 40037_SCH5390672]diacylglycerol acyltransferase/  98% 84% WP_065159015.1mycolyltransferaseAg85A [Mycobacterium asiaticum] diacylglycerolacyltransferase/ 100% 82% WP_062899503.1 mycolyltransferaseAg85A[Mycobacterium avium] MULTISPECIES: hypothetical 100% 82% WP_003876576.1protein [Mycobacterium avium complex (MAC)] diacylglycerolacyltransferase/  98% 84% WP_065044249.1 mycolyltransferase Ag85A[Mycobacterium gordonae] hypothetical protein  98% 82% WP_012394484.1[Mycobacterium marinum] secreted antigen 85-B  97% 82% BAV41604.1[Mycobacterium ulcerans subsp. shinshuense] hypothetical protein 100%81% WP_010949276.1 [Mycobacterium avium] antigen 85-B [Mycobacterium 96% 83% CQD16344.1 europaeum] hypothetical protein  98% 82%WP_007771267.1 [Mycobacterium colombiense] diacylglycerolacyltransferase  98% 82% WP_043954940.1 [Mycobacterium indicus pranii]diacylglycerol acyltransferase/ 100% 81% WP_068288134.1mycolyltransferase Ag85A [Mycobacterium sp. E2462] diacylglycerolacyltransferase  98% 81% WP_036426578.1 [Mycobacterium sp. 012931] 85Bprotein [Mycobacterium 100% 81% AAM21939.1 avium subsp.paratuberculosis] diacylglycerol acyltransferase/ 96% 82% WP_064934819.1mycolyltransferase Ag85A [Mycobacterium intracellulare] secreted antigen85-B FbpB  98% 81% ABL05230.1 [Mycobacterium ulcerans Agy99]diacylglycerol acyltransferase  98% 86% WP_036402954.1 [Mycobacteriumkansasii] Esterase [Mycobacterium  97% 81% EPQ47622.1 sp. 012931]diacylglycerol acyltransferase/  98% 81% WP_067934350.1mycolyltransferase Ag85A [Mycobacterium sp. E2479] diacylglycerolacyltransferase  98% 85% WP_036418777.1 [Mycobacterium gastri]diacylglycerol acyltransferase/  98% 81% WP_064951422.1mycolyltransferaseAg85A [Mycobacterium colombiense] diacylglycerolacyltransferase/  98% 83% WP_055380413.1 mycolyltransferaseAg85AMycobacterium tuberculosis] esterase, putative, antigen 85-B  98% 83%AAK46207.1 [Mycobacterium tuberculosis CDC1551] diacylglycerolacyltransferase/ 100% 82% WP_066912698.1 mycolyltransferase Ag85A[Mycobacterium interjectum] antigen 85-B [Mycobacterium  97% 86%ETZ99389.1 kansasii 824] antigen 85-B [Mycobacterium 100% 81% CPR05988.1bohemicum DSM 44277] diacylglycerol acyltransferase/  98% 83%WP_047713277.1 mycolyltransferase Ag85A [Mycobacterium bovis]

TABLE 3 Results of alignment of ML2055 amino acid sequence with otherspecies Name Cover Identity Accession alanine and proline-rich 96% 85%WP_045843569.1 secreted protein Apa [Mycobacterium lepromatosis] alanineand proline-rich 94% 61% AIR16824.1 secreted protein Apa [Mycobacteriumkansasii 662] alanine and proline-rich 94% 74% WP_047317005.1 secretedprotein Apa [Mycobacterium haemophilum] hypothetical protein 94% 63%WP_065145552.1 [Mycobacterium asiaticum] alanine and proline-rich 94%68% WP_031695336.1 secreted protein Apa [Mycobacterium tuberculosis]hypothetical protein 94% 61% WP_065165242.1 [Mycobacterium gordonae]hypothetical protein 94% 62% WP_068156628.1 [Mycobacterium szulgai]hypothetical protein 94% 65% WP_055369424.1 [Mycobacterium tuberculosis]hypothetical protein 94% 66% WP_067743767.1 [Mycobacterium sp. 852014-50255_SCH5639931] alanine and proline rich 60% 80% BAV41641.1 secretedprotein [Mycobacterium ulcerans subsp. shinshuense] alanine and prolinerich 60% 80% WP_015355514.1 secreted protein Apa [Mycobacteriumliflandii] alanine and proline-rich 94% 64% WP_044081122.1 secretedprotein Apa [Mycobacterium canettii] alanine and proline rich 60% 80%WP_020725275.1 secreted protein [Mycobacterium marinum] alanine andproline-rich 60% 79% WP_036417094.1 secreted protein Apa [Mycobacteriumgastri] hypothetical protein 94% 58% WP_067409357.1 [Mycobacterium sp.1423905.2] hypothetical protein 94% 66% WP_066933007.1 [Mycobacteriumsp. 1554424.7] hypothetical protein 94% 65% WP_024456921.1[Mycobacterium bovis] hypothetical protein 94% 64% WP_068079301.1[Mycobacterium sp. E1747] fibronectin attachment protein 94% 65%WP_015293251.1 [Mycobacterium canettii]

TABLE 4 Results of alignment of ML2380 amino acid sequence with otherspecies Name Cover Identity Accession hypothetical protein 100% 89%WP_045843787.1 [Mycobacterium lepromatosis] hypothetical protein 100%87% WP_047313676.1 [Mycobacterium haemophilum] hypothetical protein  99%74% WP_065144676.1 [Mycobacterium asiaticum] MULTISPECIES: hypothetical 88% 73% WP_051128635.1 protein [Mycobacterium] hypothetical protein 94% 71% WP_064985021.1 [Mycobacterium mucogenicum] hypothetical proteinTL10_07350  92% 68% KIU17456.1 [Mycobacterium llatzerense] hypotheticalprotein 100% 65% WP_040542321.1 [Mycobacterium vaccae] hypotheticalprotein  99% 65% WP_048631131.1 [Mycobacterium aurum] hypotheticalprotein  99% 67% WP_060999962.1 [Mycobacterium mucogenicum] hypotheticalprotein 100% 64% WP_067953437.1 [Mycobacterium sp. NAZ190054]hypothetical protein  95% 68% KMO66863.1 MCHLDSM_07340 [Mycobacteriumchlorophenolicum] hypothetical protein  82% 75% WP_048421400.1[Mycobacterium chubuense] hypothetical protein  82% 74% WP_057150842.1[Mycobacterium sp. Soil538] hypothetical protein  96% 64% WP_024447804.1[Mycobacterium iranicum] hypothetical protein  99% 65% WP_068289290.1[Mycobacterium sp. E2462] hypothetical protein  99% 61% WP_011894296.1[Mycobacterium gilvum] hypothetical protein  83% 75% WP_036470504.1[Mycobacterium neoaurum] hypothetical protein BN971_  99% 66% CPR11699.102987 [Mycobacterium bohemicum DSM 44277] hypothetical protein  93% 65%WP_029115183.1 [Mycobacterium sp. URHB0044] bypothetical protein  99%66% WP_031705761.1 [Mycobacterium tuberculosis] hypothetical protein 99% 66% WP_015289054.1 [Mycobacterium canettii] hypothetical protein 99% 66% WP_067256445.1 [Mycobacterium sp. 852002- 10029_SCHS224772]hypothetical protein  99% 66% WP_003910126.1 [Mycobacterium africanum]hypothetical protein MT047]  99% 66% AAK44694.1 [Mycobacteriumtuberculosis CDC1551] hypothetical protein  99% 65% WP_066959997.1[Mycobacterium sp. 852002- 50816_SCH5313054-b] hypothetical protein  95%66% WP_036421759.1 [Mycobacterium sp. 360MFTsu5.1] hypothetical protein 83% 75% WP_036462037.1 [Mycobacterium sp. UNCCL9] hypothetical protein 99% 65% CQD10070.1 BN000_02058 [Mycobacterium europaeum] hypotheticalprotein  99% 66% WP_067933987.1 [Mycobacterium sp. E2479] hypotheticalprotein  99% 66% WP_036457021.1 [Mycobacterium marinum] hypotheticalprotein  99% 65% WP_066917676.1 [Mycobacterium interjectum] hypotheticalprotein  99% 65% WP_068277017.1 [Mycobacterium sp. E787] hypotheticalprotein  99% 66% WP_064950639.1 [Mycobacterium colombiense] hypotheticalprotein  82% 71% WP_067990334.1 [Mycobacterium sp. YC-RL4] hypotheticalprotein  79% 73% WP_043414089.1 [Mycobacterium rufum] hypotheticalprotein  99% 66% WP_067171319.1 [Mycobacterium sp. 1165549.7]hypothetical protein  99% 65% WP_067109731.1 [Mycobacterium sp. 852002-51057_SCH5723018] hypothetical protein  99% 64% WP_068061777.1[Mycobacterium sp. E342] hypothetical protein  79% 72% WP_066835136.1[Mycobacterium sp. 852013- 51886_SCH5428379] hypothetical protein  99%66% WP_067837577.1 [Mycobacterium sp. E3078] hypothetical protein  99%65% WP_068227057.1 [Mycobacterium sp. E3198] hypothetical protein  99%65% WP_067009291.1 [Mycobacterium sp. 1081908.1] hypothetical proteinSHTP_  99% 65% BAV42346.1 3308 [Mycobacterium ulcerans subsp.shinsbuense] hypothetical protein  99% 66% WP_067344501.1 [Mycobacteriumsp. 1245852.3] bypothetical protein  99% 64% WP_067871707.1[Mycobacterium sp. E2699] hypothetical protein  99% 66% WP_036466491.1[Mycobacterium triplex] hypothetical protein  98% 61% WP_036340591.1[Mycobacterium aromaticivorans] hypothetical protein  99% 65%WP_067151627.1 [Mycobacterium sp. 1245805.9] conserved secreted protein 99% 66% ABL03953.1 [Mycobacterium ulcerans Agy99] hypothetical protein 87% 65% WP_011778329.1 [Mycobacterium vanbaalenii] hypothetical protein 87% 64% WP_036367359.1 [Mycobacterium austroafricanum] bypotheticalprotein  99% 65% WP_066930932.1 [Mycobacterium sp. 1554424.7]hypothetical protein  99% 64% WP_067282166.1 [Mycobacteriumscrofulaceum] hypothetical protein  99% 66% WP_025737008.1[Mycobacterium genavense] hypothetical protein  99% 66% WP_067122709.1[Mycobacterium sp. 852002- 51971_SCH5477799-a]

TABLE 5 Results of alignment of ML253.1 amino acid sequence with otherspecies Name Cover Identity Accession type VII secretion protein EsxH100% 88% WP_045843896.1 [Mycobacterium lepromatosis] type VII secretionprotein EsxH 100% 74% WP_047315908.1 [Mycobacterium haemophilum] typeVII secretion protein EsxH 100% 74% WP_036351589.1 [Mycobacteriumasiaticum] ESAT-6-like protein EsxH 100% 72% WP_003902934.1[Mycobacterium tuberculosis] type VII secretion protein EsxH 100% 73%WP_055580201.1 [Mycobacterium gordonae] type VII secretion protein EsxH100% 72% WP_062541000.1 [Mycobacterium celatum] MULTISPECIES: type VII100% 70% WP_068070284.1 secretion protein EsxH [Mycobacterium] EsaT-6like protein EsxH  98% 76% AEF34288.1 [Mycobacterium sinense] lowmolecular weight protein 100% 71% EFP48790.1 antigen 7 esxH[Mycobacterium tuberculosis SUMu010] type VII secretion protein EsxH100% 71% WP_065012511.1 [Mycobacterium kyorinense] ESAT-6-like proteinEsxH 100% 70% WP_003910092.1 [Mycobacterium africanum] type VIIsecretion protein EsxH  98% 73% WP_047318399.1 [Mycobacteriumheraklionense] ESAT-6-like protein EsxH 100% 70% WP_015288927.1[Mycobacterium canettii] ESAT-6-like protein EsxH  98% 72%WP_011740245.1 [Mycobacterium marinum] type VII secretion protein EsxH 98% 72% WP_067972689.1 [Mycobacterium sp. 8WA6] type VII secretionprotein EsxH  98% 74% WP_068918824.1 [Mycobacterium sp. djl-10] 10 kDaantigen [Mycobacterium 100% 69% EPQ44287.1 sp. 012931] type VIIsecretion protein EsxH 100% 72% WP_068102777.1 [Mycobacterium sp. E2327]ESAT-6-like protein EsxH  98% 71% WP_011891324.1 [Mycobacterium gilvum]type VII secretion protein EsxH 100% 71% WP_061558056.1 [Mycobacteriumsimiae] type VII secretion protein EsxH  98% 72% WP_066851115.1[Mycobacterium sp. 1274756.6] type VII secretion protein EsxH  98% 72%WP_024443849.1 [Mycobacterium sp. UM_WGJ] type VII secretion proteinEsxH 100% 70% WP_046187146.1 [Mycobacterium nebraskense] EsaT-6 likeprotein EsxH 100% 68% ABL03785.1 [Mycobacterium ulcerans Agy99] type VIIsecretion protein EsxH 100% 71% WP_067013661.1 [Mycobacterium sp.1081908.1] type VII secretion protein EsxH 100% 68% WP_063467440.1[Mycobacterium kansasii] type VII secretion protein EsxH 100% 69%WP_067104747.1 [Mycobacterium sp. 852002- 40037_SCH5390672] type VIIsecretion protein EsxH 100% 70% WP_068138018.1 [Mycobacterium sp. E796]type VII secretion protein EsxH 100% 70% WP_068040952.1 [Mycobacteriumsp. E2733] type VII secretion protein EsxH 100% 68% WP_066998456.1[Mycobacterium sp. 1465703.0] type VII secretion protein EsxH 100% 71%WP_067116855.1 [Mycobacterium sp. 852002- 51057_SCH5723018] type VIIsecretion protein EsxH 100% 68% WP_067413879.1 [Mycobacterium sp.1423905.2] type VII secretion protein EsxH  98% 68% WP_064281562.1[Mycobacterium iranicum] low molecular weight protein  98% 72%CPR13219.1 antigen 7 Cfp7 [Mycobacterium bohemicum DSM 44277] type VIIsecretion protein EsxH 100% 70% WP_066929865.1 [Mycobacterium sp.1554424.7] type VII secretion protein EsxH 100% 71% WP_048893951.1[Mycobacterium heckeshornense] low molecular weight protein 100% 67%ABK67570.1 antigen 7 Cfp7 [Mycobacterium avium 104] hypothetical proteinGuangZ0019_ 100% 67% EQM16518.1 4184 [Mycobacterium tuberculosisGuangZ0019] type VII secretion protein EsxH 100% 69% WP_067330420.1[Mycobacterium sp. 1245111.1]

1. A method for stimulating an immune response in a mammal comprisingadministering to a mammal in need thereof a fusion polypeptidecomprising Mycobacterium leprae (M. leprae) antigens ML2028, ML2055, andML2380, or M. leprae antigens having at least 90% amino acid sequenceidentity to ML2028, ML2055, and ML2380.
 2. The method of claim 1,wherein the fusion polypeptide further comprises M. leprae antigenML2531 or an M. leprae antigen having at least 90% amino acid sequenceidentify to ML2531.
 3. The method of claim 1, further comprisingadministering to the mammal M. bovis BCG vaccine.
 4. The method of claim1, wherein M. bovis BCG vaccine was previously administered to themammal.
 5. (canceled)
 6. (canceled)
 7. The method of claim 1, whereinthe mammal is a human healthy household contact of a human identified asbeing infected with M. leprae, the mammal has been infected by M.leprae, or the mammal exhibits signs or symptoms of infection by M.leprae.
 8. (canceled)
 9. (canceled)
 10. The method of claim 1, whereinfrom 1 ug to about 20 ug or from 1 ug to about 10 ug of the compositionis administered to the mammal per dose.
 11. (canceled)
 12. (canceled)13. A method for treating or reducing the time course of chemotherapyagainst a Mycobacterium leprae (M. leprae) infection in a mammal, themethod comprising administering to a mammal having an M. lepraeinfection a fusion polypeptide comprising M. leprae antigens ML2028,ML2055, and ML2380, or M. leprae antigens having at least 90% amino acidsequence identity to ML2028, ML2055, and ML2380.
 14. The method of claim13, wherein the fusion polypeptide further comprises M. leprae antigenML2531 or an M. leprae antigen having at least 90% amino acid sequenceidentify to ML2531.
 15. The method of claim 13, further comprisingadministering to the mammal one or more chemotherapeutic agents.
 16. Themethod of claim 15, wherein the one or more chemotherapeutic agentscomprise one or more agents selected from the group consisting ofdapsone, rifampicin, clofazimine, ofloxacin, minocycline, gatifloxacin,linezolid, and PA
 824. 17. The method of claim 15, wherein: (i) themammal is first administered one or more chemotherapeutic agents over aperiod of time and subsequently administered the composition, (ii) themammal is first administered the composition and subsequentlyadministered one or more chemotherapeutic agents over a period of time;(iii) administration of the one or more chemotherapeutic agents and thecomposition is concurrent.
 18. (canceled)
 19. (canceled)
 20. The methodof claim 13, further comprising administering the composition to themammal one or more subsequent times.
 21. The method of claim 13, furthercomprising administering to the mammal M. bovis BCG vaccine.
 22. Themethod of claim 13, wherein M. bovis BCG vaccine was previouslyadministered to the mammal. 23-29. (canceled)
 30. The method of claim13, wherein the mammal is infected with a multidrug resistant M. leprae.31-33. (canceled)
 34. The method of claim 13, wherein the time course ofchemotherapy is shortened to no more than about 3 months, about 5months, or about 7 months.
 35. (canceled)
 36. The method of claim 13,wherein from 1 ug to about 20 ug or from 1 ug to about 10 ug of thecomposition is administered to the mammal per dose.
 37. The method ofclaim 13, further comprising administering the immunostimulant GLA isadministered to the mammal.
 38. A method for detecting Mycobacteriumleprae (M. leprae) infection in a biological sample, comprising: (a)contacting a biological sample with a fusion polypeptide comprising M.leprae antigens ML2028, ML2055, and ML2380, or M. leprae antigens havingat least 90% amino acid sequence identity to ML2028, ML2055, and ML2380;and (b) detecting in the biological sample the presence of antibodiesthat bind to the fusion polypeptide, thereby detecting M. lepraeinfection in a biological sample.
 39. The method of claim 38, whereinthe fusion polypeptide further comprises M. leprae antigen ML2531 or anM. leprae antigen having at least 90% amino acid sequence identify toML2531. 40-46. (canceled)